Novel eto1 genes and use of same for reduced ethylene and improved stress tolerance in plants

ABSTRACT

The invention provides isolated ethylene over-producer 1 (ETO1) nucleic acid molecules which are associated with ethylene production in plants and their encoded proteins. The present invention provides methods and compositions relating to altering ethylene production and abiotic stress response in plants. The invention further provides recombinant expression cassettes, host cells, transgenic plants and antibody compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/850,717 filed Aug. 5, 2010 and claims the benefit of U.S. ProvisionalPatent Application No. 61/231,379, filed Aug. 5, 2009, both of which arehereby incorporated herein in their entirety by reference.

TECHNICAL FIELD

The present invention relates generally to plant molecular biology. Morespecifically, it relates to nucleic acids and methods for modulatingtheir expression in plants.

BACKGROUND OF THE INVENTION

Plant hormones have been intensively studied for decades for theirdiverse and complex effects on plant life. Of the five mainhormones-auxins, ethylene, abscisic acid, cytokinins andgibberellins-the molecular signaling and mode of action of ethylene hasbeen the most fully researched.

Ethylene (C₂H₄) is a gaseous plant hormone that affects myriaddevelopmental processes and fitness responses in plants, such asgermination, flower and leaf senescence, fruit ripening, leafabscission, root nodulation, programmed cell death and responsiveness tostress and pathogen attack. Over the past decade, genetic screens haveidentified more than a dozen genes involved in the ethylene response inplants.

Ethylene and the ethylene response pathways govern diverse processes inplants, and these effects are sometimes affected by the action of otherplant hormones, other physiological signals and the environment, bothbiotic and abiotic. For example, it is known that cytokinin can causeethylene like effects through the action of ethylene. In addition,abscisic acid can inhibit ethylene production and signaling. Auxin andethylene are also known to cooperate in various physiological phenomena.Such physiological activities of ethylene include, but are not limitedto, promotion of food ripening, abscission of leaves and fruit ofdicotyledonous species, flower senescence, stem extension of aquaticplants, gas space (aerenchyma) development in roots, leaf epinasticcurvatures, stem and shoot swelling (in association with stunting),femaleness in cucurbits, fruit growth in certain species, apical hookclosure in etiolated shoots, root hair formation, flowering in theBromeliaceae, diageotropism of etiolated shoots, and increased geneexpression (e.g., of polygalacturonase, cellulase, chitinases,β1,3-glucanases, etc.). Ethylene is released naturally by ripening fruitand is also produced by most plant tissues, e.g., in response to stress(e.g., drought, crowding, disease or pathogen attack, temperature (coldor heat) stress, wounding, etc.) and in maturing and senescing organs.

Ethylene is generated from methionine by a well-defined pathwayinvolving the conversion of S-adenosyl-L-methionine (SAM or Ado Met) tothe cyclic amino acid 1-aminocyclopropane-1-carboxylic acid (ACC) whichis facilitated by ACC synthase. ACC synthase is an aminotransferasewhich catalyzes the rate limiting step in the formation of ethylene byconverting S-adenosylmethionine to ACC.

Ethylene is then produced from the oxidation of ACC through the actionof ACC oxidase (also known as the ethylene forming enzyme) with hydrogencyanide as a secondary product that is detoxified by β-cyanoalaninesynthase. Finally, ethylene can be metabolized by oxidation to CO₂ or toethylene oxide and ethylene glycol.

There is a continuing need for modulation of ethylene production and itsresponse pathways in plants for manipulating plant development or stressresponses. This invention relates to novel ethylene over-producer 1(ETO1) sequences and their use in plants to inhibit ethylene productionby removal of a critical component on the ethylene synthesis pathway.The invention includes novel polynucleotide sequences, expressionconstructs, vectors, plant cells and resultant plants. These and otherfeatures of the invention will become apparent upon review of thefollowing materials.

SUMMARY OF THE INVENTION

This invention involves the identification and characterization of novelETO1 genes from maize and soybean which may be introduced into plants tomodulate ethylene production and improve stress tolerance in plants.ETO1 is a protein that negatively regulates ACS activity and concomitantethylene production. ACS refers to ACC synthase, where ACC is1-aminocyclopropane-1-carboxylic acid.

The invention comprises polynucleotides, related polypeptides and allconservatively modified variants of the maize and soybean ETO1 sequencespresented herein.

The invention also includes methods to alter the genetic composition ofcrop plants, especially maize and soybean, so that such crops can bemore tolerant to abiotic stress conditions and to modulate otherethylene mediated responses. The utility of this class of invention isthen both yield enhancement and stress tolerance.

Ethylene-mediated responses include but are not limited to thoseinvolving: crowding tolerance, seed set and development, growth incompacted soils, flooding tolerance, maturation and senescence, droughttolerance and disease resistance. This invention provides methods andcompositions to effect various alterations in the ethylene-mediatedresponse in a plant that would result in improved agronomic performance,particularly under stress.

Therefore, in one aspect, the present invention relates to an isolatednucleic acid molecule comprising an isolated polynucleotide sequenceencoding an ETO1 protein which will bind to the C-terminus of ACS6 andtarget the molecule for degradation. One embodiment of the invention isan isolated polynucleotide comprising a nucleotide sequence selectedfrom the group consisting of: (a) the nucleotide sequence comprising SEQID NO: 1, 3, 5, 7 or 9; (b) the nucleotide sequence encoding an aminoacid sequence comprising SEQ ID NO: 2, 4, 6, 8 or 10; (c) apolynucleotide having a specified sequence identity to a polynucleotideencoding a polypeptide of the present invention; (d) a polynucleotidewhich is complementary to the polynucleotide of (a) and (e) apolynucleotide comprising a specified number of contiguous nucleotidesfrom a polynucleotide of (a) or (b). The isolated nucleic acid moleculecan be DNA.

Compositions of the invention include an isolated polypeptide comprisingan amino acid sequence selected from the group consisting of: (a) SEQ IDNO: 2, 4, 6, 8 or 10 and (b) the amino acid sequence comprising aspecified sequence identity to SEQ ID NO: 2, 4, 6, 8 or 10, wherein saidpolypeptide has ETO1 activity.

In another aspect, the present invention relates to a recombinantexpression cassette comprising a nucleic acid molecule as described.Additionally, the present invention relates to a vector containing therecombinant expression cassette. Further, the vector containing therecombinant expression cassette can facilitate the transcription andtranslation of the nucleic acid molecule in a host cell. The presentinvention also relates to the host cells able to express thepolynucleotide of the present invention. A number of host cells could beused, such as but not limited to, microbial, mammalian, plant or insect.Preferably, the host cells are non-human host cells.

In yet another embodiment, the present invention is directed to atransgenic plant or plant cell, containing the nucleic acid molecules ofthe present invention. Preferred plants containing the polynucleotidesof the present invention include but are not limited to maize, soybean,sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, tomatoand millet. In another embodiment, the transgenic plant is a maize plantor plant cell. Another embodiment is the transgenic seeds from thetransgenic plant.

The plants of the invention can have altered ethyleneproduction/response as compared to a control plant. In some plants, thealtered ethylene production/response is directed to a vegetative tissue,a reproductive tissue or a vegetative tissue and a reproductive tissue.Plants of the invention can have at least one of the followingphenotypes including but not limited to: differences in crowdingtolerance, seed set and development, growth in compacted soils, floodingtolerance, drought tolerance, maturation and senescence and diseaseresistance, compared to non transformed plants.

Methods for decreasing ethylene synthesis in a plant are provided byintroducing to the same an ETO1 protein, thereby targeting ACS6 fordegradation and removing it from the ethylene synthesis pathway. Themethod can comprise introducing into the plant an ETO1 polynucleotide ofthe invention.

In a further aspect, the present invention relates to a polynucleotideamplified from a Zea mays or Glycine max nucleic acid library usingprimers which selectively hybridize, under stringent hybridizationconditions, to loci within polynucleotides of the present invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Units, prefixes and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges recitedwithin the specification are inclusive of the numbers defining the rangeand include each integer within the defined range. Amino acids may bereferred to herein by either their commonly known three letter symbolsor by the one-letter symbols recommended by the IUPAC-IUB BiochemicalNomenclature Commission. Nucleotides, likewise, may be referred to bytheir commonly accepted single-letter codes. Unless otherwise providedfor, software, electrical, and electronics terms as used herein are asdefined in The New IEEE Standard Dictionary of Electrical andElectronics Terms (5th edition, 1993). The terms defined below are morefully defined by reference to the specification as a whole.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology Principles and Applications, Persing, et al., Ed., AmericanSociety for Microbiology, Washington, D.C. (1993). The product ofamplification is termed an amplicon.

The term “antibody” includes reference to antigen binding forms ofantibodies (e.g., Faba, F (ab) 2). The term “antibody” frequently refersto a polypeptide substantially encoded by an immunoglobulin gene orimmunoglobulin genes or fragments thereof which specifically bind andrecognize an analyte (antigen). However, while various antibodyfragments can be defined in terms of the digestion of an intactantibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by utilizing recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments such as single chain FV, chimeric antibodies (i.e.,comprising constant and variable regions from different species),humanized antibodies (i.e., comprising a complementarity determiningregion (CDR) from a non-human source) and heteroconjugate antibodies(e.g., bispecific antibodies).

The term “antigen” includes reference to a substance to which anantibody can be generated and/or to which the antibody is specificallyimmunoreactive. The specific immunoreactive sites within the antigen areknown as epitopes or antigenic determinants. These epitopes can be alinear array of monomers in a polymeric composition-such as amino acidsin a protein- or consist of or comprise a more complex secondary ortertiary structure. Those of skill will recognize that all immunogens(i.e., substances capable of eliciting an immune response) are antigens;however some antigens, such as haptens, are not immunogens but may bemade immunogenic by coupling to a carrier molecule. An antibodyimmunologically reactive with a particular antigen can be generated invivo or by recombinant methods such as selection of libraries ofrecombinant antibodies in phage or similar vectors. See, e.g., Huse, etal., (1989) Science 246:1275-1281 and Ward, et al., (1989) Nature341:544-546 and Vaughan, et al., (1996) Nature Biotech. 14:309-314.

As used herein, “antisense orientation” includes reference to a duplexpolynucleotide sequence that is operably linked to a promoter in anorientation where the antisense strand is transcribed. The antisensestrand is sufficiently complementary to an endogenous transcriptionproduct such that translation of the endogenous transcription product isoften inhibited.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or conservatively modified variants of theamino acid sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenprotein. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations” and represent onespecies of conservatively modified variation. Every nucleic acidsequence herein that encodes a polypeptide also, by reference to thegenetic code, describes every possible silent variation of the nucleicacid.

One of ordinary skill will recognize that each codon in a nucleic acid(except AUG, which is ordinarily the only codon for methionine and UGG,which is ordinarily the only codon for tryptophan) can be modified toyield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide of the presentinvention is implicit in each described polypeptide sequence and iswithin the scope of the present invention.

As to amino acid sequences, one of skill will recognize that individualsubstitution, deletion or addition to a nucleic acid, peptide,polypeptide or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceresults in a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Thus, any number of amino acid residues selected from thegroup of integers consisting of from 1 to 15 can be so altered. Thus,for example, 1, 2, 3, 4, 5, 7 or 10 alterations can be made.

Conservatively modified variants typically provide similar biologicalactivity as the unmodified polypeptide sequence from which they arederived. For example, substrate specificity, enzyme activity, orligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%,80% or 90% of the native protein for its native substrate. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See also,        Creighton (1984) Proteins W. H. Freeman and Company.

By “encoding” or “encoded”, with respect to a specified nucleic acidmolecule, is meant comprising the information for translation into thespecified protein. A nucleic acid molecule encoding a protein maycomprise intervening sequences (e.g., introns) within translated regionsof the nucleic acid molecule, or may lack such interveningnon-translated sequences (e.g., as in cDNA). The information by which aprotein is encoded is specified by the use of codons. Typically, theamino acid sequence is encoded by the nucleic acid molecule using the“universal” genetic code. However, variants of the universal code, suchas are present in some plant, animal and fungal mitochondria, thebacterium Mycoplasma capricolumn or the ciliate Macronucleus, may beused when the nucleic acid is expressed therein. When the nucleic acidis prepared or altered synthetically, advantage can be taken of knowncodon preferences of the intended host where the nucleic acid is to beexpressed.

For example, although nucleic acid sequences of the present inventionmay be expressed in both monocotyledonous and dicotyledonous plantspecies, sequences can be modified to account for the specific codonpreferences and GC content preferences of monocotyledons or dicotyledonsas these preferences have been shown to differ (Murray, et al., (1989)Nucl. Acids Res. 17:477-498). Thus, the maize preferred codon for aparticular amino acid may be derived from known gene sequences frommaize. Maize codon usage for 28 genes from maize plants is listed inTable 4 of Murray, et al., supra.

As used herein “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire amino acidsequence of, a native (nonsynthetic), endogenous, biologically activeform of the specified protein. Methods to determine whether a sequenceis full-length are well known in the art including such exemplarytechniques as northern or western blots, primer extension, S 1protection, and ribonuclease protection. See, e.g., Plant MolecularBiology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin(1997). Comparison to known full-length homologous (orthologous and/orparalogous) sequences can also be used to identify full-length sequencesof the present invention. Additionally, consensus sequences typicallypresent at the 5′ and 3′ untranslated regions of mRNA aid in theidentification of a polynucleotide as full-length. For example, theconsensus sequence ANNNNAUGG, where the AUG codon therein represents theN-terminal methionine, aids in determining whether the polynucleotidehas a complete 5′ end. Consensus sequences at the 3′ end, such aspolyadenylation sequences, aid in determining whether the polynucleotidehas a complete 3′ end.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived, or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

By “host cell” is meant a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as E. coli or eukaryotic cells such as yeast,insect, amphibian or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells. A particularly preferredmonocotyledonous host cell is a maize host cell.

The term “hybridization complex” includes reference to a duplex nucleicacid structure formed by two single-stranded nucleic acid sequencesselectively hybridized with each other.

By “immunologically reactive conditions” or “immunoreactive conditions”is meant conditions which allow an antibody, reactive to a particularepitope, to bind to that epitope to a detectably greater degree (e.g.,at least 2-fold over background) than the antibody binds tosubstantially any other epitopes in a reaction mixture comprising theparticular epitope. Immunologically reactive conditions are dependentupon the format of the antibody binding reaction and typically are thoseutilized in immunoassay protocols. See Harlow and Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York (1988), fora description of immunoassay formats and conditions.

The term “introduced” in the context of inserting a nucleic acidmolecule into a cell, means “transfection” or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid molecule into a eukaryotic or prokaryotic cell where the nucleicacid molecule may be incorporated into the genome of the cell (e.g.,chromosome, plasmid, plastid or mitochondrial DNA), converted into anautonomous replicon or transiently expressed (e.g., transfected mRNA).

The term “isolated” refers to material, such as a nucleic acid moleculeor a protein, which is: (1) substantially or essentially free fromcomponents that normally accompany or interact with it as found in itsnaturally occurring environment. The isolated material optionallycomprises material not found with the material in its naturalenvironment; or (2) if the material is in its natural environment, thematerial has been synthetically (non-naturally) altered by deliberatehuman intervention to a composition and/or placed at a location in thecell (e.g., genome or subcellular organelle) not native to a materialfound in that environment. The alteration to yield the syntheticmaterial can be performed on the material within or removed from itsnatural state. For example, a naturally occurring nucleic acid moleculebecomes an isolated nucleic acid molecule if it is altered, or if it istranscribed from DNA which has been altered, by means of humanintervention performed within the cell from which it originates. See,e.g., Compounds and Methods for Site Directed Mutagenesis in EukaryoticCells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo Homologous SequenceTargeting in Eukaryotic Cells; Zarling et al., PCT/US93/03868. Likewise,a naturally occurring nucleic acid molecule (e.g., a promoter) becomesisolated if it is introduced by normaturally occurring means to a locusof the genome not native to that nucleic acid molecule. Nucleic acidmolecules which are “isolated” as defined herein are also referred to as“heterologous” nucleic acid molecules.

Unless otherwise stated, the term “ETO1 nucleic acid” (also referred toherein as “ETO1 nucleic acid molecule”) is a nucleic acid (also referredto herein as a “nucleic acid molecule”) of the present invention andmeans a nucleic acid comprising a polynucleotide of the presentinvention (an “ETO1 polynucleotide”) encoding an ETO1 polypeptide withETO1 activity. An “ETO1 gene” is a gene of the present invention andrefers to a heterologous genomic form of a full-length ETO1polynucleotide.

A “subject plant or plant cell” is one in which genetic alteration, suchas transformation, has been affected as to a gene of interest or is aplant or plant cell which is descended from a plant or cell so alteredand which comprises the alteration. A “control” or “control plant” or“control plant cell” provides a reference point for measuring changes inphenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-typeplant or cell, i.e., of the same genotype as the starting material forthe genetic alteration which resulted in the subject plant or cell; (b)a plant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e., with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell genetically identical to the subjectplant or plant cell but which is not exposed to conditions or stimulithat would induce expression of the gene of interest; or (e) the subjectplant or plant cell itself, under conditions in which the gene ofinterest is not expressed.

As used herein, “localized within the chromosomal region defined by andincluding” with respect to particular markers includes reference to acontiguous length of a chromosome delimited by and including the statedmarkers.

As used herein, “marker” includes reference to a locus on a chromosomethat serves to identify a unique position on the chromosome. A“polymorphic marker” includes reference to a marker which appears inmultiple forms (alleles) such that different forms of the marker, whenthey are present in a homologous pair, allow transmission of each of thechromosomes of that pair to be followed. A genotype may be defined byuse of one or a plurality of markers.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or RNAmolecules which comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism. Constructionof exemplary nucleic acid libraries, such as genomic and cDNA libraries,is taught in standard molecular biology references such as Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology,Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook, etal., Molecular Cloning-A Laboratory Manual, 2nd ed., Vol. 1-3 (1989);and Current Protocols in Molecular Biology, Ausubel, et al., Eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc. (1994).

As used herein “operably linked” includes reference to a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. Generally, operably linked meansthat the nucleic acid sequences being linked are contiguous and wherenecessary to join two protein coding regions, contiguous and in the samereading frame.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds and progeny ofsame. Plant cell, as used herein includes, without limitation, a cellderived from a seed, suspension culture, embryo, meristematic region,callus tissue, leaf, root, shoot, gametophyte, sporophyte, pollen ormicrospore. The class of plants which can be used in the methods of theinvention is generally as broad as the class of higher plants amenableto transformation techniques, including both monocotyledonous anddicotyledonous plants. A particularly preferred plant is Zea mays.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide or analogs thereof that havethe essential nature of a natural ribonucleotide in that they hybridize,under stringent hybridization conditions, to substantially the samenucleotide sequence as naturally occurring nucleotides and/or allowtranslation into the same amino acid(s) as the naturally occurringnucleotide(s). A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art.

The term polynucleotide as it is employed herein embraces suchchemically, enzymatically or metabolically modified forms ofpolynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation. It will be appreciated, as is wellknown and as noted above, that polypeptides are not always entirelylinear. For instance, polypeptides may be branched as a result ofubiquitization, and they may be circular, with or without branching,generally as a result of post translation events, including naturalprocessing event and events brought about by human manipulation which donot occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translation natural process andby entirely synthetic methods, as well. Further, this inventioncontemplates the use of both the methionine-containing and themethionine-less amino terminal variants of the protein of the invention.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells whether or not its origin is a plant cell. Exemplary plantpromoters include, but are not limited to, those that are obtained fromplants, plant viruses, and bacteria which comprise genes expressed inplant cells such as Agrobacterium or Rhizobium. Examples of promotersunder developmental control include promoters that preferentiallyinitiate transcription in certain tissues, such as leaves, roots, orseeds. Such promoters are referred to as “tissue preferred”. Promoterswhich initiate transcription only in certain tissue are referred to as“tissue specific”. A “cell type” preferred promoter primarily drivesexpression in certain cell types in one or more organs, for example,vascular cells in roots or leaves. An “inducible” or “repressible”promoter is a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light. Tissuespecific, tissue preferred, cell type preferred, and inducible promotersare members of the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions and/or in most tissues of a plant and/or atmost developmental stages.

The term “ETO1 polypeptide” refers to a polypeptide of the presentinvention which has ETO1 activity and refers to one or more amino acidsequences, in glycosylated or non-glycosylated form. The term is alsoinclusive of fragments, variants, homologs, alleles or precursors (e.g.,preproproteins or proproteins) thereof which retain activity. An “ETO1protein” is a protein of the present invention and comprises an ETO1polypeptide. “ETO1 activity” means that the polypeptide is capable ofbinding to the C-terminus of ACS Class II enzymes and ushering them tothe 26S proteasome for degradation resulting in a decrease in ethyleneproduction as measurable by any of a number of available protocols.

As used herein “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found in identicalform within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under-expressed ornot expressed at all, as a result of deliberate human intervention. Theterm “recombinant” as used herein does not encompass the alteration ofthe cell or vector by naturally occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without deliberate human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements which permit transcription of aparticular nucleic acid in a host cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed and apromoter.

As used herein, a chimeric gene comprises a coding sequence operablylinked to a transcription initiation region that is heterologous to thecoding sequence.

The terms “residue” and “amino acid residue” and “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and unless otherwiselimited, may encompass non-natural analogs of natural amino acids thatcan function in a similar manner as naturally occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toas other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to an analyte having the recognized epitope toa substantially greater degree (e.g., at least 2-fold over background)than to substantially all analytes lacking the epitope which are presentin the sample. Specific binding to an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular protein. For example, antibodies raised to the polypeptidesof the present invention can be selected from to obtain antibodiesspecifically reactive with polypeptides of the present invention. Theproteins used as immunogens can be in native conformation or denaturedso as to provide a linear epitope.

A variety of immunoassay formats may be used to select antibodiesspecifically reactive with a particular protein (or other analyte). Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. See,Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York (1988), for a description of immunoassay formatsand conditions that can be used to determine selective reactivity.

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing).

Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, optionally less than 500 nucleotides inlength.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 MNaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.and a wash in 1× to 2×SSC (20×SSC=3.0 MNaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 MNaCl, 1% SDS at 37° C., and awash in <RTI 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 MNaCl, 1% SDS at37° C. and a wash in 0.1×SSC at 60 to 65° C. Specificity is typicallythe function of post-hybridization washes, the critical factors beingthe ionic strength and temperature of the final wash solution. ForDNA/DNA hybrids, the Tm can be approximated from the equation ofMeinkoth and Wahl, (1984) Anal. Biochem., 138:267-284:T_(m)=81.5°C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarityof monovalent cations, % GC is the percentage of guanosine and cytosinenucleotides in the DNA, % form is the percentage of formamide in thehybridization solution, and L is the length of the hybrid in base pairs.The Tm is the temperature (under defined ionic strength and pH) at which50% of a complementary target sequence hybridizes to a perfectly matchedprobe. Tm is reduced by about 1° C. for each 1% of mismatching; thus,Tm, hybridization and/or wash conditions can be adjusted to hybridize tosequences of the desired identity. For example, if sequences with >90%identity are sought, the Tm can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3 or 4° C. lower than thethermal melting point (Tm); moderately stringent conditions can utilizea hybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than thethermal melting point (Tm); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than thethermal melting point (Tm). Using the equation, hybridization and washcompositions, and desired Tm, those of ordinary skill will understandthat variations in the stringency of hybridization and/or wash solutionsare inherently described. If the desired degree of mismatching resultsin a Tm of less than 45° C. (aqueous solution) or 32° C. (formamidesolution) it is preferred to increase the SSC concentration so that ahigher temperature can be used. An extensive guide to the hybridizationof nucleic acids is found in Tijssen, Laboratory Techniques inBiochemistry and Molecular Biology-Hybridization with Nucleic AcidProbes, Part I, Chapter 2 “Overview of principles of hybridization andthe strategy of nucleic acid probe assays”, Elsevier, New York (1993);and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995).

As used herein, “transgenic plant” includes reference to a plant whichcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomesuch that the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization, nonrecombinant viral infection, non-recombinant bacterial transformation,non-recombinant transposition or spontaneous mutation.

As used herein, “vector” includes reference to a nucleic acid used intransfection of a host cell and into which can be inserted apolynucleotide. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationshipsbetween a polynucleotide/polypeptide of the present invention with areference polynucleotide/polypeptide: (a) “reference sequence”, (b)“comparison window”, (c) “sequence identity” and (d) “percentage ofsequence identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison with a polynucleotide/polypeptide of thepresent invention. A reference sequence may be a subset or the entiretyof a specified sequence; for example, as a segment of a full-length cDNAor gene sequence or the complete cDNA or gene sequence.

(b) As used herein, “comparison window” includes reference to acontiguous and specified segment of a polynucleotide/polypeptidesequence, wherein the polynucleotide/polypeptide sequence may becompared to a reference sequence and wherein the portion of thepolynucleotide/polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Generally, the comparison window is atleast 20 contiguous nucleotides/amino acids residues in length andoptionally can be 30, 40, 50, 100 or longer. Those of skill in the artunderstand that to avoid a high similarity to a reference sequence dueto inclusion of gaps in the polynucleotide/polypeptide sequence, a gappenalty is typically introduced and is subtracted from the number ofmatches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, (1981) Adv. Appl.Math. 2:482; by the homology alignment algorithm of Needleman andWunsch, (1970) J. Mol. Biol. 48:443; by the search for similarity methodof Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. 85:2444; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA and TFASTA in the GCG WisconsinGenetics Software Package, Version 10 (available from Accelrys Inc.,9685 Scranton Road, San Diego, Calif., USA). The CLUSTAL program is welldescribed by Higgins and Sharp, (1988) Gene 73:237-244; Higgins andSharp, (1989) CABIOS 5:151-153; Corpet, et al., (1988) Nucleic AcidsResearch 16:10881-90; Huang, et al., (1992) Computer Applications in theBiosciences 8:155-65 and Pearson, et al., (1994) Methods in MolecularBiology 24:307-331.

The BLAST family of programs which can be used for database similaritysearches includes: BLASTN for nucleotide query sequences againstnucleotide database sequences; BLASTX for nucleotide query sequencesagainst protein database sequences; BLASTP for protein query sequencesagainst protein database sequences; TBLASTN for protein query sequencesagainst nucleotide database sequences and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, Current Protocolsin Molecular Biology, Chapter 19, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995); Altschul et al.,(1990) J. Mol. Biol., 215:403-410 and Altschul, et al., (1997) NucleicAcids Res. 25:3389-3402.

Software for performing BLAST analyses is publicly available, e.g.,through the National Center for Biotechnology Information. Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold. These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always>0) and N (penalty score for mismatchingresidues; always<0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score.

Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a word length (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a word length (W) of 3, an expectation (E) of 10 andthe BLOSUM62 scoring matrix (see, Henikoff and Henikoff, (1989) Proc.Natl. Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul, (1993) Proc. Nat'l. Acad.Sci. USA 90:5873-5877). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. BLAST searches assume thatproteins can be modeled as random sequences. However, many real proteinscomprise regions of nonrandom sequences which may be homopolymerictracts, short-period repeats or regions enriched in one or more aminoacids. Such low-complexity regions may be aligned between unrelatedproteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, (1993) Comput. Chem., 17:149-163) and XNU (Clayerie andStates, (1993) Comput. Chem., 17:191-201) low-complexity filters can beemployed alone or in combination.

Unless otherwise stated, nucleotide and protein identity/similarityvalues provided herein are calculated using GAP (GCG Version 10) underdefault values. GAP (Global Alignment Program) can also be used tocompare a polynucleotide or polypeptide of the present invention with areference sequence. GAP uses the algorithm of Needleman and Wunsch (J.Mol. Biol. 48:443-453 (1970)) to find the alignment of two completesequences that maximizes the number of matches and minimizes the numberof gaps. GAP considers all possible alignments and gap positions andcreates the alignment with the largest number of matched bases and thefewest gaps. It allows for the provision of a gap creation penalty and agap extension penalty in units of matched bases. GAP must make a profitof gap creation penalty number of matches for each gap it inserts. If agap extension penalty greater than zero is chosen, GAP must, inaddition, make a profit for each gap inserted of the length of the gaptimes the gap extension penalty. Default gap creation penalty values andgap extension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 100. Thus, for example, the gapcreation and gap extension penalties can each independently be: 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity and Similarity. The Quality is the metric maximized in order toalign the sequences. Ratio is the quality divided by the number of basesin the shorter segment. Percent Identity is the percent of the symbolsthat actually match. Percent Similarity is the percent of the symbolsthat are similar. Symbols that are across from gaps are ignored. Asimilarity is scored when the scoring matrix value for a pair of symbolsis greater than or equal to 0.50, the similarity threshold. The scoringmatrix used in Version 10 of the Wisconsin Genetics Software Package isBLOSUM62 (see, Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA89:10915).

Multiple alignment of the sequences can be performed using the CLUSTALmethod of alignment (Higgins and Sharp, (1989) CABIOS 5:151-153) withthe default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the CLUSTAL method are KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences includes reference to theresidues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller, (1988) Computer Applic. Biol. Sci.,4:11-17, e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

Overview

The present invention provides, among other things, compositions andmethods for modulating (i.e., increasing or decreasing) the level ofpolynucleotides and polypeptides of the present invention in plants. Inparticular, the polynucleotides and polypeptides of the presentinvention can be expressed temporally or spatially, e.g., atdevelopmental stages, in tissues, and/or in quantities, which areuncharacteristic of non-recombinantly engineered plants. Thus, thepresent invention provides utility in such exemplary applications asprovided below.

Applicants have isolated a novel ETO1 protein that may be used in themodulation of ethylene activity and production in plants. The novelprotein, its nucleotide sequences encoding the same and resultantconstructs, vectors and modified plant cells, tissues, seeds and organsform the basis of the invention. The enzyme thus finds utility in anumber of stress response applications such as the following.

Crowding Tolerance

The agronomic performance of crop plants is often a function of how wellthey tolerate planting density. Overcrowded plants grow poorly. Thestress of overcrowding can be due to simple limitations of nutrients,water and sunlight. Crowding stress may also be due to enhanced contactbetween plants. Plants often respond to physical contact by slowinggrowth and thickening their tissues.

Ethylene has been implicated in plant crowding response. For example,ethylene insensitive tobacco plants did not slow growth when contactingneighboring plants (Knoester, et al., (1998) PNAS USA 95:1933-1937).There is also evidence that ethylene, and the plant's response to it isinvolved in water deficit stress and that ethylene may be causingchanges in the plant that limit its growth and aggravate the symptoms ofdrought stress beyond the loss of water itself.

The present invention provides for decreasing ethylene production in aplant, in particular cereals such as maize, by providing one or morenovel ETO1 polynucleotides or their protein products to promotetolerance of close spacing with reduced stress and yield loss.

Seed Set and Development in Maize

Ethylene plays a number of roles in seed development. For example, inmaize ethylene is linked to programmed cell death of developingendosperm cells (Young, et al., (1997) Plant Physiol., 115:737-751). Inaddition, ethylene is linked to kernel abortion, such as occurs at thetips of ears, especially in plants grown under stressful conditions(Cheng and Lur, (1996) Physiol. Plant 98:245-252). Reduced kernel seedset is of course a contributor to reduced yields. Consequently, thepresent invention provides plants, in particular maize plants, that havereduced ethylene action by providing for and/or modulating theexpression/activity of the novel ETO1 polynucleotides of the invention.

Growth in Compacted Soils

Plant growth is affected by the density and compaction of soils. Denser,more compacted soils typically result in poorer plant growth. The trendin agriculture towards more minimal till planting and cultivationpractices, with the goal of soil and energy conservation, is increasingthe need for crop plants that can perform well under these conditions.

Ethylene is well-known to affect plant growth and development and oneeffect of ethylene is to promote tissue thickening and growthretardation when encountering mechanical stress, such as compactedsoils. This can affect both the roots and shoots. This effect ispresumably adaptive in some circumstances in that it results instronger, more compact tissues that can force their way through oraround, obstacles such as compacted soils. However, in such conditions,the production of ethylene and the activation of the ethylene pathwaymay exceed what is needed for adaptive accommodation to the mechanicalstress of the compacted soils. And, of course, any resulting unnecessarygrowth inhibition would be an undesired agronomic result.

The present invention provides for decreasing ethylene production in aplant, in particular cereals such as maize, by providing for and/ormodulating the expression/activity of one or more novel ETO1polynucleotides or their protein products. Such modulated plants growand germinate better in compacted soils, resulting in higher standcounts, the herald of higher yields.

Flooding Tolerance

Flooding and water-logged soils cause substantial losses in crop yieldeach year around the world. Flooding can be both widespread or local,transitory or prolonged. Ethylene has been implicated in floodingmediated damage. In fact, in flooded conditions ethylene production canrise. There are two main reasons for this rise: 1) under such floodedconditions, which creates hypoxia, plants produce more ethylene and 2)under flooded conditions the diffusion of ethylene away from the plantis slowed, because ethylene is minimally soluble in water, resulting ina rise of intra-plant ethylene levels.

Ethylene in flooded maize roots can also inhibit gravitropism, which isnormally adaptive during germination in that it orients the roots downand the shoots up. Gravitropism is a factor in determining rootarchitecture, which in turn plays an important role in soil resourceacquisition. Manipulation of ethylene levels could be used to impactroot angle for drought tolerance, flood tolerance, greater standabilityand/or improved nutrient uptake. For example, a root growing at a moreerect angle (steeper) would likely grow more deeply in soil and thusobtain water at greater depths, improving drought tolerance. In theabsence of drought stress a converse argument could be made for moreefficient root uptake of nutrients and water in the upper layers of thesoil profile, by roots which are more parallel to the soil surface. Ingeneral, roots that have a angle nearer that of vertical (steep) arealso more susceptible to root lodging than roots with a shallow angle(parallel to the surface) that can be more root lodging resistant.

In addition to inhibition of gravitropism, it is likely that ethyleneevolution in flooded conditions inhibits growth, especially of roots.Such inhibition will likely contribute to poor plant growth overall, andconsequently is a disadvantageous agronomic trait.

The present invention provides for decreasing ethylene production in aplant, in particular cereals such as maize, by providing for and/ormodulating the expression/activity of one or more novel ETO1polynucleotides or their protein products. Such plants should grow andgerminate better in flooded conditions or water-logged soils, resultingin higher stand counts.

Plant Maturation and Senescence

Ethylene is known to be involved in controlling senescence, fruitripening and abscission.

The role of ethylene in fruit ripening is well-established andindustrially applied. The prediction based on precedent would be thatethylene underproduction/insensitivity would result in slower seedripening and the converse would result in more rapid seed ripening.Abscission is primarily studied for dicot plants and apparently haslittle application to monocots such as cereals. Ethylene mediatedsenescence also is mostly studied in dicots, but control of senescenceis agronomically important for both dicot and monocot crop species.Ethylene insensitivity can cause a delay of, but not arrest, senescence.The senescence process mediated by ethylene bears some similarities tothe cell death process in disease symptoms and in abscission zones.

Controlling ethylene production, as through the control of one or morenovel ETO1 genes, could result in modulation of maturity rates for cropplants such as maize.

The present invention provides for decreasing ethylene production in aplant, in particular cereals such as maize, by providing for and/ormodulating the expression/activity of one or more novel ETO1polynucleotides or their protein products which may contribute to alater maturing plant, which is desirable for placing crop varieties indifferent maturity zones.

Tolerance to Other Abiotic Stresses

Many stresses on plants induce the production of ethylene (see, Morganand Drew, (1997) Physiol. Plant 100:620-630). These stresses can becold, heat, wounding, pollution, drought and hypersalinity. Mechanicalimpedance (soil compaction) and flooding stresses were addressed above.It appears that several of these stresses operate through commonmechanisms, such as water deficit. Clearly drought causes water deficit;crowding stress may also cause water deficit. Additionally, in maizechilling can cause an elevation in ethylene production and activity, andthis induction is apparently due to chilling causing water deficit incells (Janowaik and Dorffling, (1995) J. Plant Physiol. 147:257-262).

Some of the ethylene production following stresses may serve an adaptivepurpose by regulating ethylene-mediated processes in the plant thatresult in a plant reorganized in such manner to better acclimate to thestress encountered. However, there is also evidence that ethyleneproduction during stress can result in an aggravation of negativesymptoms resulting from the stress, such as yellowing, tissue death andsenescence.

To the extent that ethylene production during stress causes or augmentsnegative stress-related symptoms, it would be desirable to create a cropplant with reduced ethylene production. Towards that end, the presentinvention provides for decreasing ethylene production in a plant, inparticular cereals such as maize, by providing for and/or modulating theexpression/activity of one or more novel ETO1 polynucleotides or theirprotein products to create plants that avoid certain ethylene-mediatedeffects.

Disease Resistance

Crop plants can be susceptible to a wide variety of pathogens, whetherviruses, bacteria, fungi or insects. This susceptibility results inlarge crop yield losses annually worldwide. Crop breeders haveendeavored to breed more resistant or tolerant varieties which canwithstand pathogen attack. Additional genetic engineering strategiesseek the same end. In many plant-pathogen interactions the symptoms ofdisease, most often tissue necrosis and resulting poor plant growth, areknown to be the result of an active plant defense response to thepathogen. That is, the symptoms are caused directly by the plant and notsimply by the pathogen. From among the list of all crop plants and theirpotential list of pathogens, resistance is the rule, and susceptibilitythe exception. Susceptible interactions are often thought to result froman improper or insufficient activation defense by the plant that resultsin a runaway symptom development and an inability to contain thepathogen.

Ethylene has long been known to be associated with plant pathogendefense systems. Many pathogenesis related genes are induced inexpression at the level of mRNA by ethylene. The trend in ourunderstanding of the role of ethylene in plant pathogen defense istowards ethylene and ethylene mediated effects being viewed asprincipally part of the downstream reactions to pathogen attack, as insymptom development. Ethylene seems to be involved in the plant'sresponse to the stress of pathogen attack and in tissue damage inflictedby the pathogen. In a susceptible interaction ethylene may actuallypromote tissue damage. Consequently, in such situations, blockingethylene production or action may actually result in less tissue damage,that is, more apparent resistance, even though the pathogen iscompatible with the plant. Blocking ethylene action is known to resultin either more susceptibility (e.g., Knoester, et al., (1988)) or moreresistance (e.g., Lund, et al., (1998) Plant Cell 10:371-382), whichindicates that the role of ethylene action is complex, as is to beexpected, for it depends upon the interactions of diverse plants andpathogens.

The present invention provides for the use of one or more novel ETO1polynucleotides or their protein products to affect enhanced resistanceto plant stresses, in particular for monocots such as maize.

For most applications this will involve the reduction in ethyleneproduction by providing for and/or modulating the expression/activity ofnovel ETO1 polynucleotides or their proteins, with the goal of causingplants that produce less ethylene in response to stress and therebyplants that are less prone to tissue damage following exposure toabiotic stressors. ETO1 is a potent negative regulator of ACS andethylene production, see, for example, Chae and Faure, et al., (2003)Plant Cell 15(2):545-559; Christians, et al., (2009) The Plant Journal57(2):332-345; Wang, et al., (2004) Nature 428(6986):945-950; Yoshida,et al., (2005) BMC Plant Biology 5:14 and Yoshida, et al., (2006) PlantMolecular Biology 62(3):427-437.

Plant Transformation

The generation of transgenic plants is central to crop plant geneticengineering strategies. Transgenesis typically involves the introductionof exogenous DNA into the plants cells via a variety of methods, such asparticle bombardment or Agrobacterium infection, which is usuallyfollowed by tissue culture and plant regeneration. Transgenic plantproduction remains a costly and rate limiting step in geneticengineering, especially for many of the most economically important cropplants, such as the cereals, like maize.

Improving the Efficiency of this Process is Therefore of GreatImportance.

It has been accepted for a long time that ethylene action has negativeconsequences for plant transformation. As a result various approaches tobind, trap or otherwise block the accumulation of ethylene are employedin transformation and tissue culture (see, Songstad, et al., (1991)Plant Cell Reports 9:694-702). The particle bombardment method causessubstantial tissue/cell damage and such damage is known to elicitethylene accumulation. Moreover, in most tissue culture methods, sometissue grows better than others, as is designed in chemical selection oftransformants. Such dying tissue can emit ethylene and cause inhibitionof positive transformants. Aggravating these effects is the confinementof plant tissues in containers for the purpose of tissue regenerationthat can result in the accumulation of ethylene, also causing growthretardation. As ethylene is known to promote slower tissue growth andeven cell/tissue death, having a means to block or minimize ethyleneaction during transformation is desired.

Consequently, the present invention also provides for use of the ETO1sequences herein to create transient or stable reductions in ethyleneaction by increasing the expression/activity of ETO1 polynucleotides orpolypeptides.

Other Utilities

The present invention also provides isolated nucleic acids comprisingpolynucleotides of sufficient length and complementarity to a gene ofthe present invention to use as probes or amplification primers in thedetection, quantitation or isolation of gene transcripts. For example,isolated nucleic acids of the present invention can be used as probes indetecting deficiencies in the level of mRNA in screenings for desiredtransgenic plants, for detecting mutations in the gene (e.g.,substitutions, deletions or additions), for monitoring upregulation ofexpression or changes in enzyme activity in screening assays ofcompounds, for detection of any number of allelic variants(polymorphisms), orthologs or paralogs of the gene, or for site directedmutagenesis in eukaryotic cells (see, e.g., U.S. Pat. No. 5,565,350).The isolated nucleic acids of the present invention can also be used forrecombinant expression of their encoded polypeptides, or for use asimmunogens in the preparation and/or screening of antibodies. Theisolated nucleic acids of the present invention can also be employed foruse in sense or antisense suppression of one or more genes of thepresent invention in a host cell, tissue or plant. Attachment ofchemical agents which bind, intercalate, cleave and/or cross-link to theisolated nucleic acids of the present invention can also be used tomodulate transcription or translation.

The present invention also provides isolated proteins comprising apolypeptide of the present invention (e.g., preproenzyme, proenzyme orenzymes). The present invention also provides proteins comprising atleast one epitope from a polypeptide of the present invention. Theproteins of the present invention can be employed in assays for enzymeagonists or antagonists of enzyme function, or for use as immunogens orantigens to obtain antibodies specifically immunoreactive with a proteinof the present invention. Such antibodies can be used in assays forexpression levels, for identifying and/or isolating nucleic acids of thepresent invention from expression libraries, for identification ofhomologous polypeptides from other species or for purification ofpolypeptides of the present invention.

The isolated nucleic acids and polypeptides of the present invention canbe used over a broad range of plant types, particularly monocots such asthe species of the family Gramineae including Hordeum, Secale, Tritium,Sorghum (e.g., S. bicolor) and Zea (e.g., Z. mays). The isolated nucleicacid and proteins of the present invention can also be used in speciesfrom the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus,Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum,Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis,Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum,Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus,Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum,Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia,Glycine, Pisum, Phaseolus, Lolium, Oryza and Avena.

Nucleic Acids

The present invention provides, among other things, isolated nucleicacids of RNA, DNA and analogs and/or chimeras thereof, comprising apolynucleotide of the present invention.

A polynucleotide of the present invention is inclusive of:

-   -   (a) a polynucleotide encoding a polypeptide of SEQ ID NO: 2, 4,        6, 8 or 10 and conservatively modified and polymorphic variants        thereof, including exemplary polynucleotides of SEQ ID NO: 1, 3,        5, 7 or 9, (ETO1);    -   (b) an isolated polynucleotide which is the product of        amplification from a plant nucleic acid library using primer        pairs which selectively hybridize under stringent conditions to        loci within a polynucleotide of the present invention;    -   (c) an isolated polynucleotide which selectively hybridizes to a        polynucleotide of (a) or (b);    -   (d) an isolated polynucleotide having a specified sequence        identity with polynucleotides of (a), (b) or (c);    -   (e) an isolated polynucleotide encoding a protein having a        specified number of contiguous amino acids from a prototype        polypeptide, wherein the protein is specifically recognized by        antisera elicited by presentation of the protein and wherein the        protein does not detectably immunoreact to antisera which has        been fully immunosorbed with the protein;    -   (f) complementary sequences of polynucleotides of (a), (b),        (c), (d) or (e); and    -   (g) an isolated polynucleotide comprising at least a specific        number of contiguous nucleotides from a polynucleotide of (a),        (b), (c), (d), (e) or (f);    -   (h) an isolated polynucleotide from a full-length enriched cDNA        library having the physico-chemical property of selectively        hybridizing to a polynucleotide of (a), (b), (c), (d), (e), (f)        or (g);    -   (i) an isolated polynucleotide made by the process of: 1)        providing a full-length enriched nucleic acid library, 2)        selectively hybridizing the polynucleotide to a polynucleotide        of (a), (b), (c), (d), (e), (f), (g) or (h), thereby isolating        the polynucleotide from the nucleic acid library.

A. Polynucleotides Encoding a Polypeptide of the Present Invention

As indicated in (a), above, the present invention provides isolatednucleic acids comprising a polynucleotide of the present invention,wherein the polynucleotide encodes a polypeptide of the presentinvention. Every nucleic acid sequence herein that encodes a polypeptidealso, by reference to the genetic code, describes every possible silentvariation of the nucleic acid. One of ordinary skill will recognize thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for methionine and UGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Thus, each silent variation of a nucleic acid which encodes apolypeptide of the present invention is implicit in each describedpolypeptide sequence and is within the scope of the present invention.Accordingly, the present invention includes polynucleotides of thepresent invention and polynucleotides encoding a polypeptide of thepresent invention.

B. Polynucleotides Amplified from a Plant Nucleic Acid Library

As indicated in (b), above, the present invention provides an isolatednucleic acid comprising a polynucleotide of the present invention,wherein the polynucleotides are amplified, under nucleic acidamplification conditions, from a plant nucleic acid library.

Nucleic acid amplification conditions for each of the variety ofamplification methods are well known to those of ordinary skill in theart. The plant nucleic acid library can be constructed from a monocotsuch as a cereal crop. Exemplary cereals include corn, sorghum, alfalfa,canola, wheat or rice. The plant nucleic acid library can also beconstructed from a dicot such as soybean. Zea mays lines B73, PHRE1,A632, BMP2#10, W23 and Mol7 are known and publicly available. Otherpublicly known and available maize lines can be obtained from the MaizeGenetics Cooperation (Urbana, Ill.).

Wheat lines are available from the Wheat Genetics Resource Center(Manhattan, Kans.). The nucleic acid library may be a cDNA library, agenomic library or a library generally constructed from nucleartranscripts at any stage of intron processing. cDNA libraries can benormalized to increase the representation of relatively rare cDNAs. Inoptional embodiments, the cDNA library is constructed using an enrichedfull-length cDNA synthesis method. Examples of such methods includeOligo-Capping (Maruyama and Sugano, (1994) Gene 138:171-174),Biotinylated CAP Trapper (Carninci, et al., (1996) Genomics 37:327-336)and CAP Retention Procedure (Edery, et al., (1995) Molecular andCellular Biology 15:3363-3371). Rapidly growing tissues or rapidlydividing cells are preferred for use as an mRNA source for constructionof a cDNA library. Growth stages of corn are described in “How a CornPlant Develops, “Special Report No. 48, Iowa State University of Scienceand Technology Cooperative Extension Service, Ames, Iowa, ReprintedFebruary 1993.

A polynucleotide of this embodiment (or subsequences thereof) can beobtained, for example, by using amplification primers which areselectively hybridized and primer extended, under nucleic acidamplification conditions, to at least two sites within a polynucleotideof the present invention or to two sites within the nucleic acid whichflank and comprise a polynucleotide of the present invention or to asite within a polynucleotide of the present invention and a site withinthe nucleic acid which comprises it. Methods for obtaining 5′ and/or 3′ends of a vector insert are well known in the art. See, e.g., RACE(Rapid Amplification of Complementary Ends) as described in Frohman, inPCR Protocols: A Guide to Methods and Applications, Innis, et al., Eds.(Academic Press, Inc., San Diego), pp. 28-38 (1990)), see also, U.S.Pat. No. 5,470,722, and Current Protocols in Molecular Biology, Unit15.6, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience,New York (1995); Frohman and Martin, (1989) Techniques 1:165.

Optionally, the primers are complementary to a subsequence of the targetnucleic acid which they amplify but may have a sequence identity rangingfrom about 85% to 99% relative to the polynucleotide sequence which theyare designed to anneal to. As those skilled in the art will appreciate,the sites to which the primer pairs will selectively hybridize arechosen such that a single contiguous nucleic acid can be formed underthe desired nucleic acid amplification conditions. The primer length innucleotides is selected from the group of integers consisting of from atleast 15 to 50. Thus, the primers can be at least 15, 18, 20, 25, 30, 40or 50 nucleotides in length. Those of skill will recognize that alengthened primer sequence can be employed to increase specificity ofbinding (i.e., annealing) to a target sequence. A non-annealing sequenceat the 5′ end of a primer (a “tail”) can be added, for example, tointroduce a cloning site at the terminal ends of the amplicon.

The amplification products can be translated using expression systemswell known to those of skill in the art. The resulting translationproducts can be confirmed as polypeptides of the present invention by,for example, assaying for the appropriate catalytic activity (e.g.,specific activity and/or substrate specificity) or verifying thepresence of one or more epitopes which are specific to a polypeptide ofthe present invention. Methods for protein synthesis from PCR derivedtemplates are known in the art and available commercially. See, e.g.,Amersham Life Sciences, Inc, Catalog '97, p. 354.

C. Polynucleotides which Selectively Hybridize to a Polynucleotide of(A) or (B)

As indicated in (c), above, the present invention provides isolatednucleic acids comprising polynucleotides of the present invention,wherein the polynucleotides selectively hybridize, under selectivehybridization conditions, to a polynucleotide of sections (A) or (B) asdiscussed above. Thus, the polynucleotides of this embodiment can beused for isolating, detecting, and/or quantifying nucleic acidscomprising the polynucleotides of (A) or (B). For example,polynucleotides of the present invention can be used to identify,isolate or amplify partial or full-length clones in a deposited library.

In some embodiments, the polynucleotides are genomic or cDNA sequencesisolated or otherwise complementary to a cDNA from a dicot or monocotnucleic acid library.

Exemplary species of monocots and dicots include, but are not limitedto: maize, canola, soybean, cotton, wheat, sorghum, sunflower, alfalfa,oats, sugar cane, millet, barley and rice. The cDNA library comprises atleast 50% to 95% full-length sequences (for example, at least 50%, 60%,70%, 80%, 90% or 95% full-length sequences). The cDNA libraries can benormalized to increase the representation of rare sequences. See, e.g.,U.S. Pat. No. 5,482,845. Low stringency hybridization conditions aretypically, but not exclusively, employed with sequences having a reducedsequence identity relative to complementary sequences. Moderate and highstringency conditions can optionally be employed for sequences ofgreater identity. Low stringency conditions allow selectivehybridization of sequences having about 70% to 80% sequence identity andcan be employed to identify orthologous or paralogous sequences.

D. Polynucleotides Having a Specific Sequence Identity with thePolynucleotides of (A), (B) or (C)

As indicated in (d), above, the present invention provides isolatednucleic acids comprising polynucleotides of the present invention,wherein the polynucleotides have a specified identity at the nucleotidelevel to a polynucleotide as disclosed above in sections (A), (B), or(C), above. Identity can be calculated using, for example, the BLAST,CLUSTALW or GAP algorithms under default conditions. The percentage ofidentity to a reference sequence is at least 60% and rounded upwards tothe nearest integer, can be expressed as an integer selected from thegroup of integers consisting of from 60 to 99. Thus, for example, thepercentage of identity to a reference sequence can be at least 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% to afull-length sequence of the invention.

Optionally, the polynucleotides of this embodiment will encode apolypeptide that will share an epitope with a polypeptide encoded by thepolynucleotides of sections (A), (B) or (C). Thus, these polynucleotidesencode a first polypeptide which elicits production of antiseracomprising antibodies which are specifically reactive to a secondpolypeptide encoded by a polynucleotide of (A), (B) or (C). However, thefirst polypeptide does not bind to antisera raised against itself whenthe antisera has been fully immunosorbed with the first polypeptide.Hence, the polynucleotides of this embodiment can be used to generateantibodies for use in, for example, the screening of expressionlibraries for nucleic acids comprising polynucleotides of (A), (B) or(C), or for purification of, or in immunoassays for, polypeptidesencoded by the polynucleotides of (A), (B) or (C). The polynucleotidesof this embodiment comprise nucleic acid sequences which can be employedfor selective hybridization to a polynucleotide encoding a polypeptideof the present invention.

Screening polypeptides for specific binding to antisera can beconveniently achieved using peptide display libraries. This methodinvolves the screening of large collections of peptides for individualmembers having the desired function or structure.

Antibody screening of peptide display libraries is well known in theart. The displayed peptide sequences can be from 3 to 5000 or more aminoacids in length, frequently from 5100 amino acids long, and often fromabout 8 to 15 amino acids long. In addition to direct chemical syntheticmethods for generating peptide libraries, several recombinant DNAmethods have been described. One type involves the display of a peptidesequence on the surface of a bacteriophage or cell. Each bacteriophageor cell contains the nucleotide sequence encoding the particulardisplayed peptide sequence. Such methods are described in PCT PatentApplication Publication Numbers 1991/17271, 1991/18980, 1991/19818 and1993/08278. Other systems for generating libraries of peptides haveaspects of both in vitro chemical synthesis and recombinant methods.See, PCT Patent Application Publication Numbers 1992/05258, 1992/14843and 1997/20078. See also, U.S. Pat. Nos. 5,658,754 and 5,643,768.Peptide display libraries, vectors and screening kits are commerciallyavailable from such suppliers as Invitrogen (Carlsbad, Calif.).

E. Polynucleotides Encoding a Protein Having a Subsequence from aPrototype Polypeptide and Cross-Reactive to the Prototype Polypeptide

As indicated in (e), above, the present invention provides isolatednucleic acids comprising polynucleotides of the present invention,wherein the polynucleotides encode a protein having a subsequence ofcontiguous amino acids from a prototype polypeptide of the presentinvention such as are provided in (a), above. The length of contiguousamino acids from the prototype polypeptide is selected from the group ofintegers consisting of from at least 10 to the number of amino acidswithin the prototype sequence. Thus, for example, the polynucleotide canencode a polypeptide having a subsequence having at least 10, 15, 20,25, 30, 35, 40, 45 or 50, contiguous amino acids from the prototypepolypeptide. Further, the number of such subsequences encoded by apolynucleotide of the instant embodiment can be any integer selectedfrom the group consisting of from 1 to 20, such as 2, 3, 4 or 5. Thesubsequences can be separated by any integer of nucleotides from 1 tothe number of nucleotides in the sequence such as at least 5, 10, 15,25, 50, 100 or 200 nucleotides.

The proteins encoded by polynucleotides of this embodiment, whenpresented as an immunogen, elicit the production of polyclonalantibodies which specifically bind to a prototype polypeptide such asbut not limited to, a polypeptide encoded by the polynucleotide of (a)or (b), above. Generally, however, a protein encoded by a polynucleotideof this embodiment does not bind to antisera raised against theprototype polypeptide when the antisera has been fully immunosorbed withthe prototype polypeptide. Methods of making and assaying for antibodybinding specificity/affinity are well known in the art. Exemplaryimmunoassay formats include ELISA, competitive immunoassays,radioimmunoassays, Western blots, indirect immunofluorescent assays andthe like.

In a preferred assay method, fully immunosorbed and pooled antiserawhich is elicited to the prototype polypeptide can be used in acompetitive binding assay to test the protein. The concentration of theprototype polypeptide required to inhibit 50% of the binding of theantisera to the prototype polypeptide is determined. If the amount ofthe protein required to inhibit binding is less than twice the amount ofthe prototype protein, then the protein is said to specifically bind tothe antisera elicited to the immunogen.

Accordingly, the proteins of the present invention embrace allelicvariants, conservatively modified variants and minor recombinantmodifications to a prototype polypeptide.

A polynucleotide of the present invention optionally encodes a proteinhaving a molecular weight as the non-glycosylated protein within 20% ofthe molecular weight of the full-length non-glycosylated polypeptides ofthe present invention. Molecular weight can be readily determined bySDS-PAGE under reducing conditions. Optionally, the molecular weight iswithin 15% of a full length polypeptide of the present invention, morepreferably within 10% or 5% and most preferably within 3%, 2% or 1% of afull length polypeptide of the present invention. Optionally, thepolynucleotides of this embodiment will encode a protein having aspecific enzymatic activity at least 50%, 60%, 80% or 90% of a cellularextract comprising the native, endogenous full-length polypeptide of thepresent invention.

Further, the proteins encoded by polynucleotides of this embodiment willoptionally have a substantially similar affinity constant (Km) and/orcatalytic activity (i.e., the microscopic rate constant, kcat) as thenative endogenous, full-length protein. Those of skill in the art willrecognize that kcat/Km value determines the specificity for competingsubstrates and is often referred to as the specificity constant.Proteins of this embodiment can have akcat/Km value at least 10% of afull-length polypeptide of the present invention as determined using theendogenous substrate of that polypeptide. Optionally, the kcat/Km valuewill be at least 20%, 30%, 40%, 50% and most preferably at least 60%,70%, 80%, 90% or 95% the kcat/Km value of the full-length polypeptide ofthe present invention.

Determination of kcat, Km and kcat/Km can be determined by any number ofmeans well known to those of skill in the art. For example, the initialrates (i.e., the first 5% or less of the reaction) can be determinedusing rapid mixing and sampling techniques (e.g., continuous-flow,stopped-flow or rapid quenching techniques), flash photolysis orrelaxation methods (e.g., temperature jumps) in conjunction with suchexemplary methods of measuring as spectrophotometry, spectrofluorimetry,nuclear magnetic resonance or radioactive procedures. Kinetic values areconveniently obtained using a Lineweaver Burk or Eadie-Hofstee plot.

F. Polynucleotides Complementary to the Polynucleotides of (A)-(E)

As indicated in (f), above, the present invention provides isolatednucleic acids comprising polynucleotides complementary to thepolynucleotides of paragraphs A-E, above. As those of skill in the artwill recognize, complementary sequences base-pair throughout theentirety of their length with the polynucleotides of sections (A)-(E)(i.e., have 100% sequence identity over their entire length).Complementary bases associate through hydrogen bonding in doublestranded nucleic acids. For example, the following base pairs arecomplementary: guanine and cytosine; adenine and thymine and adenine anduracil.

G. Polynucleotides which are Subsequences of the Polynucleotides of(A)-(F)

As indicated in (g), above, the present invention provides isolatednucleic acids comprising polynucleotides which comprise at least 15contiguous bases from the polynucleotides of sections (A) through (F) asdiscussed above. The length of the polynucleotide is given as an integerselected from the group consisting of from at least 15 to the length ofthe nucleic acid sequence from which the polynucleotide is a subsequenceof. Thus, for example, polynucleotides of the present invention areinclusive of polynucleotides comprising at least 15, 20, 25, 30, 40, 50,60, 75 or 100 contiguous nucleotides in length from the polynucleotidesof (A)-(F). Optionally, the number of such subsequences encoded by apolynucleotide of the instant embodiment can be any integer selectedfrom the group consisting of from 1 to 20, such as 2, 3, 4 or 5. Thesubsequences can be separated by any integer of nucleotides from 1 tothe number of nucleotides in the sequence such as at least 5, 10, 15,25, 50, 100 or 200 nucleotides.

Subsequences can be made by in vitro synthetic, in vitro biosynthetic orin vivo recombinant methods. In optional embodiments, subsequences canbe made by nucleic acid amplification. For example, nucleic acid primerswill be constructed to selectively hybridize to a sequence (or itscomplement) within, or co-extensive with, the coding region.

The subsequences of the present invention can comprise structurallibraries as are known in the art and discussed briefly below. The cDNAlibrary comprises at least 50% to 95% full-length sequences (forexample, at least 50%, 60%, 70%, 80%, 90% or 95% full-length sequences).The cDNA library can be constructed from a variety of tissues from amonocot or dicot at a variety of developmental stages. Exemplary speciesinclude maize, wheat, rice, canola, soybean, cotton, sorghum, sunflower,alfalfa, oats, sugar cane, millet, barley and rice. Methods ofselectively hybridizing, under selective hybridization conditions, apolynucleotide from a full-length enriched library to a polynucleotideof the present invention are known to those of ordinary skill in theart. Any number of stringency conditions can be employed to allow forselective hybridization. In optional embodiments, the stringency allowsfor selective hybridization of sequences having at least 70%, 75%, 80%,85%, 90%, 95% or 98% sequence identity over the length of the hybridizedregion. Full-length enriched cDNA libraries can be normalized toincrease the representation of rare sequences.

H. Polynucleotide Products Made by a cDNA Isolation Process

As indicated in (I), above, the present invention provides an isolatedpolynucleotide made by the process of: 1) providing a full-lengthenriched nucleic acid library, 2) selectively hybridizing thepolynucleotide to a polynucleotide of paragraphs (A), (B), (C), (D),(E), (F), (G) or (H) as discussed above and thereby isolating thepolynucleotide from the nucleic acid library. Full-length enrichednucleic acid libraries are constructed as discussed in paragraph (G) andbelow. Selective hybridization conditions are as discussed in paragraph(G). Nucleic acid purification procedures are well known in the art.

Purification can be conveniently accomplished using solid-phase methods;such methods are well known to those of skill in the art and kits areavailable from commercial suppliers such as Advanced Biotechnologies(Surrey, UK). For example, a polynucleotide of paragraphs (A)-(H) can beimmobilized to a solid support such as a membrane, bead or particle.See, e.g., U.S. Pat. No. 5,667,976. The polynucleotide product of thepresent process is selectively hybridized to an immobilizedpolynucleotide and the solid support is subsequently isolated fromnon-hybridized polynucleotides by methods including, but not limited to,centrifugation, magnetic separation, filtration, electrophoresis, andthe like.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) standard recombinant methods, (b) synthetic techniques orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention will be cloned, amplified or otherwise constructedfrom a monocot such as corn, rice or wheat or a dicot such as soybean.

The nucleic acids may conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites may beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexahistidine marker sequence provides a convenient meansto purify the proteins of the present invention. A polynucleotide of thepresent invention can be attached to a vector, adapter or linker forcloning and/or expression of a polynucleotide of the present invention.Additional sequences may be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide or to improve the introduction ofthe polynucleotide into a cell. Typically, the length of a nucleic acidof the present invention less the length of its polynucleotide of thepresent invention is less than 20 kilobase pairs, often less than 15 kb,and frequently less than 10 kb. Use of cloning vectors, expressionvectors, adapters, and linkers is well known and extensively describedin the art. For a description of various nucleic acids see, for example,Stratagene Cloning Systems, Catalogs 1999 (La Jolla, Calif.); andAmersham Life Sciences, Inc, Catalog '99 (Arlington Heights, Ill.).

A. Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA or a hybrid thereof, can be obtained from plantbiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probeswhich selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. Isolation of RNA andconstruction of cDNA and genomic libraries is well known to those ofordinary skill in the art. See, e.g., Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); andCurrent Protocols in Molecular Biology, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995).

A1. Full-Length Enriched cDNA Libraries

A number of cDNA synthesis protocols have been described which provideenriched full-length cDNA libraries. Enriched full-length cDNA librariesare constructed to comprise at least 60%, and more preferably at least70%, 80%, 90% or 95% full-length inserts amongst clones containinginserts. The length of insert in such libraries can be at least 2, 3, 4,5, 6, 7, 8, 9, 10 or more kilobase pairs. Vectors to accommodate insertsof these sizes are known in the art and available commercially. See,e.g., Stratagene's lambda ZAP Express (cDNA cloning vector with 0 to 12kb cloning capacity). An exemplary method of constructing a greater than95% pure full-length cDNA library is described by Carninci, et al.,(1996) Genomics 37:327-336. Other methods for producing full-lengthlibraries are known in the art. See, e.g., Edery, et al., (1995) Mol.Cell. Biol. 15(6):3363-3371 and PCT Application Publication Number WO1996/34981.

A2. Normalized or Subtracted cDNA Libraries

A non-normalized cDNA library represents the mRNA population of thetissue it was made from. Since unique clones are out-numbered by clonesderived from highly expressed genes their isolation can be laborious.Normalization of a cDNA library is the process of creating a library inwhich each clone is more equally represented. Construction of normalizedlibraries is described in Ko, (1990) Nucl. Acids. Res. 18(19):5705-5711;Patanjali, et al., (1991) Proc. Natl. Acad. USA 88:1943-1947; U.S. Pat.Nos. 5,482,685, 5,482,845 and 5,637,685. In an exemplary methoddescribed by Soares, et al., normalization resulted in reduction of theabundance of clones from a range of four orders of magnitude to a narrowrange of only 1 order of magnitude. Proc. Natl. Acad. Sci. USA91:9228-9232 (1994).

Subtracted cDNA libraries are another means to increase the proportionof less abundant cDNA species. In this procedure, cDNA prepared from onepool of mRNA is depleted of sequences present in a second pool of mRNAby hybridization. The cDNA: mRNA hybrids are removed and the remainingun-hybridized cDNA pool is enriched for sequences unique to that pool.See, Foote, et al. in, Plant Molecular Biology: A Laboratory Manual,Clark, Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, (1991)Technique 3(2):58-63; Sive and St. John, (1988) Nucl. Acids Res.16(22):10937; Current Protocols in Molecular Biology, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995) andSwaroop, et al., (1991) Nucl. Acids Res. 19(8):1954. cDNA subtractionkits are commercially available. See, e.g., PCR-Select (Clontech, PaloAlto, Calif.).

To construct genomic libraries, large segments of genomic DNA aregenerated by fragmentation, e.g., using restriction endonucleases, andare ligated with vector DNA to form concatemers that can be packagedinto the appropriate vector. Methodologies to accomplish these ends andsequencing methods to verify the sequence of nucleic acids are wellknown in the art. Examples of appropriate molecular biologicaltechniques and instructions sufficient to direct persons of skillthrough many construction, cloning, and screening methodologies arefound in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Vols. 1-3 (1989), Methods inEnzymology, Vol. 152: Guide to Molecular Cloning Techniques, Berger andKimmel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocolsin Molecular Biology, Ausubel, et al., Eds., Greene Publishing andWiley-Interscience, New York (1995); Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Kits forconstruction of genomic libraries are also commercially available.

The cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention such as thosedisclosed herein. Probes may be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentplant species. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay andeither the hybridization or the wash medium can be stringent.

The nucleic acids of interest can also be amplified from nucleic acidsamples using amplification techniques. For instance, polymerase chainreaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related genes directly fromgenomic DNA or cDNA libraries. PCR and other in vitro amplificationmethods may also be useful, for example, to clone nucleic acid sequencesthat code for proteins to be expressed, to make nucleic acids to use asprobes for detecting the presence of the desired mRNA in samples, fornucleic acid sequencing, or for other purposes. The T4 gene 32 protein(Boehringer Mannheim) can be used to improve yield of long PCR products.

PCR-based screening methods have been described. Wilfinger, et al.,describe a PCR-based method in which the longest cDNA is identified inthe first step so that incomplete clones can be eliminated from study.Bio Techniques 22(3):481-486 (1997). Such methods are particularlyeffective in combination with a full-length cDNA constructionmethodology, above.

B. Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by methods such as the phosphotriestermethod of Narang, et al., (1979) Meth. Enzymol. 68:90-99; thephosphodiester method of Brown, et al., (1979) Meth. Enzymol.68:109-151; the diethylphosphoramidite method of Beaucage, et al.,(1981) Tetra. Lett. 22:1859-1862; the solid phase phosphoramiditetriester method described by Beaucage and Caruthers, (1981) Tetra.Letts. 22(20):1859-1862, e.g., using an automated synthesizer, e.g., asdescribed in Needham-VanDevanter, et al., (1984) Nucleic Acids Res.12:6159-6168 and the solid support method of U.S. Pat. No. 4,458,066.Chemical synthesis generally produces a single stranded oligonucleotide.This may be converted into double stranded DNA by hybridization with acomplementary sequence or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill will recognize that whilechemical synthesis of DNA is best employed for sequences of about 100bases or less, longer sequences may be obtained by the ligation ofshorter sequences.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence coding for the desired polypeptide of the present invention,for example a cDNA or a genomic sequence encoding a full lengthpolypeptide of the present invention, can be used to construct arecombinant expression cassette which can be introduced into the desiredhost cell. A recombinant expression cassette will typically comprise apolynucleotide of the present invention operably linked totranscriptional initiation regulatory sequences which will direct thetranscription of the polynucleotide in the intended host cell, such astissues of a transformed plant.

For example, plant expression vectors may include (1) a cloned plantgene under the transcriptional control of 5′ and 3′ regulatory sequencesand (2) a dominant selectable marker. Such plant expression vectors mayalso contain, if desired, a promoter regulatory region (e.g., oneconferring inducible or constitutive, environmentally- ordevelopmentally-regulated or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site and/ora polyadenylation signal.

A plant promoter fragment can be employed which will direct expressionof a polynucleotide of the present invention in all tissues of aregenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the cauliflower mosaic virus (CaMV)³⁵S transcription initiation region, the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smaspromoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No.5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter,the GRP1-8 promoter and other transcription initiation regions fromvarious plant genes known to those of skill. Constitutive promoters ofparticular interest for use in soybean include SCP1 and At-UBQ10.

Examples of promoters under developmental control include promoters thatinitiate transcription only, or preferentially, in certain tissues, suchas leaves, roots, fruit, seeds, or flowers. Exemplary promoters includethe anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and5,689,051), glob-1 promoter and gamma-zein promoter.

Promoters of interest in constructs designed to drive expressionpreferentially in female reproductive tissues include the maize Zag2.1promoter (GenBank Number X80206; Schmidt, et al., (1993) Plant Cell5(7):729-737); maize Zap promoter (U.S. Pat. No. 7,560,612); maizeckx1-2 promoter (US Patent Application Publication Number 2002/0152500A1); ZM-ADF4 (US Patent Application Publication Number 2009/0094713);maize eep1 promoter (US Patent Application Publication Number2004/0237147); maize end2 promoter, (U.S. Pat. Nos. 6,528,704 and6,903,205); maize lec1 promoter (U.S. Pat. No. 7,122,658); maize F3.7promoter (Baszczynski, et al., (1997) Maydica 42:189-201); maize tb1promoter (Hubbarda, et al., (2002) Genetics 162:1927-1935); maize eep2promoter (US Patent Application Publication Number 2004/0237147); maizethioredoxinH promoter, US Provisional Patent Application Ser. No.60/514,123); maize Zm40 promoter (U.S. Pat. No. 6,403,862) maize mLIP15promoter (U.S. Pat. No. 6,479,734); maize ESR promoter (U.S. Pat. No.7,276,596); maize PCNA2 promoter (US Patent Application PublicationNumber 2005/0120404).

Root-preferred promoters include Zm-NAS2 (U.S. patent application Ser.No. 12/030,455, filed Feb. 13, 2008), Zm-Cyclo1 promoter (U.S. Pat. No.7,268,226), Zm-Metallothionein promoters (U.S. Pat. Nos. 6,774,282;7,214,854 and 7,214,855 (also known as RootMET2)), Zm-MSY promoter (SEQID NO: 64; U.S. Patent Application Ser. No. 60/971,310 filed Sep. 11,2007) or MsZRP promoter (SEQ ID NO: 65; see, U.S. Pat. No. 5,633,363).Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759-772); rolBpromoter (Capana, et al., (1994) Plant Mol. Biol. 25(4):681-691; and theCRWAQ81 root-preferred promoter with the ADH first intron (US PatentApplication Publication Number 2005/0097633). See also, U.S. Pat. Nos.5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732 and5,023,179.

Alternatively, the plant promoter may be under more preciseenvironmental control. Such promoters are referred to here as“inducible” promoters. Environmental conditions that may effecttranscription by inducible promoters include pathogen attack, anaerobicconditions, or the presence of light. Examples of inducible promotersare the Adhl promoter which is inducible by hypoxia or cold stress, theHsp70 promoter which is inducible by heat stress and the PPDK promoterwhich is inducible by light.

The operation of a promoter may also vary depending on its location inthe genome. Thus, an inducible promoter may become fully or partiallyconstitutive in certain locations.

Both heterologous and non-heterologous (i.e., endogenous) promoters canbe employed to direct expression of the nucleic acids of the presentinvention. These promoters can also be used, for example, in recombinantexpression cassettes to drive expression of antisense nucleic acids toreduce, increase, or alter concentration and/or composition of theproteins of the present invention in a desired tissue. Thus, in someembodiments, the nucleic acid construct will comprise a promoterfunctional in a plant cell, such as in Zea mays, operably linked to apolynucleotide of the present invention. Promoters useful in theseembodiments include the endogenous promoters driving expression of apolypeptide of the present invention.

In some embodiments, isolated nucleic acids which serve as promoter orenhancer elements can be introduced in the appropriate position(generally upstream) of a non-heterologous form of a polynucleotide ofthe present invention so as to up or down regulate expression of apolynucleotide of the present invention. For example, endogenouspromoters can be altered in vivo by mutation, deletion, and/orsubstitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCTPatent Application Number PCT/US93/03868) or isolated promoters can beintroduced into a plant cell in the proper orientation and distance froma gene of the present invention so as to control the expression of thegene. Gene expression can be modulated under conditions suitable forplant growth so as to alter the total concentration and/or alter thecomposition of the polypeptides of the present invention in plant cell.

Thus, the present invention provides compositions, and methods formaking, heterologous promoters and/or enhancers operably linked to anative, endogenous (i.e., nonheterologous) form of a polynucleotide ofthe present invention.

Methods for identifying promoters with a particular expression pattern,in terms of, e.g., tissue type, cell type, stage of development and/orenvironmental conditions, are well known in the art. See, e.g., TheMaize Handbook, Chapters 114-115, Freeling and Walbot, Eds., Springer,New York (1994); Corn and Corn Improvement, 3rd edition, Chapter 6,Sprague and Dudley, Eds., American Society of Agronomy, Madison, Wis.(1988).

A typical step in promoter isolation methods is identification of geneproducts that are expressed with some degree of specificity in thetarget tissue. Amongst the range of methodologies are: differentialhybridization to cDNA libraries; subtractive hybridization; differentialdisplay; differential 2-D protein gel electrophoresis; DNA probe arraysand isolation of proteins known to be expressed with some specificity inthe target tissue. Such methods are well known to those of skill in theart. Commercially available products for identifying promoters are knownin the art such as Clontech's (Palo Alto, Calif.) Universal GenomeWalker Kit.

For the protein-based methods, it is helpful to obtain the amino acidsequence for at least a portion of the identified protein, and then touse the protein sequence as the basis for preparing a nucleic acid thatcan be used as a probe to identify either genomic DNA directly, orpreferably, to identify a cDNA clone from a library prepared from thetarget tissue. Once such a cDNA clone has been identified, that sequencecan be used to identify the sequence at the 5′ end of the transcript ofthe indicated gene. For differential hybridization, subtractivehybridization and differential display, the nucleic acid sequenceidentified as enriched in the target tissue is used to identify thesequence at the 5′ end of the transcript of the indicated gene. Oncesuch sequences are identified, starting either from protein sequences ornucleic acid sequences, any of these sequences identified as being fromthe gene transcript can be used to screen a genomic library preparedfrom the target organism. Methods for identifying and confirming thetranscriptional start site are well known in the art.

In the process of isolating promoters expressed under particularenvironmental conditions or stresses, or in specific tissues, or atparticular developmental stages, a number of genes are identified thatare expressed under the desired circumstances, in the desired tissue, orat the desired stage. Further analysis will reveal expression of eachparticular gene in one or more other tissues of the plant. One canidentify a promoter with activity in the desired tissue or condition butthat does not have activity in any other common tissue.

To identify the promoter sequence, the 5′ portions of the clonesdescribed here are analyzed for sequences characteristic of promotersequences. For instance, promoter sequence elements include the TATA boxconsensus sequence (TATAAT), which is usually an AT-rich stretch of 5-10bp located approximately 20 to 40 base pairs upstream of thetranscription start site. Identification of the TATA box is well knownin the art. For example, one way to predict the location of this elementis to identify the transcription start site using standard RNA-mappingtechniques such as primer extension, S 1 analysis, and/or RNaseprotection. To confirm the presence of the AT-rich sequence, astructure-function analysis can be performed involving mutagenesis ofthe putative region and quantification of the mutation's effect onexpression of a linked downstream reporter gene. See, e.g., The MaizeHandbook, Chapter 114, Freeling and Walbot, Eds., Springer, New York,(1994).

In plants, further upstream from the TATA box, at positions-80 to -100,there is typically a promoter element (i.e., the CAAT box) with a seriesof adenines surrounding the trinucleotide G (or T) N G. Messing, et al.,in Genetic Engineering in Plants, Kosage, Meredith and Hollaender, Eds.,pp. 221-227 1983. In maize, there is no well conserved CAAT box butthere are several short, conserved protein-binding motifs upstream ofthe TATA box. These include motifs for the trans-acting transcriptionfactors involved in light regulation, anaerobic induction, hormonalregulation or anthocyanin biosynthesis, as appropriate for each gene.

Once promoter and/or gene sequences are known, a region of suitable sizeis selected from the genomic DNA that is 5′ to the transcriptionalstart, or the translational start site and such sequences are thenlinked to a coding sequence. If the transcriptional start site is usedas the point of fusion, any of a number of possible 5′ untranslatedregions can be used in between the transcriptional start site and thepartial coding sequence. If the translational start site at the 3′ endof the specific promoter is used, then it is linked directly to themethionine start codon of a coding sequence.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes or alternatively from another plantgene or less preferably from any other eukaryotic gene.

An intron sequence can be added to the 5′ untranslated region or thecoding sequence of the partial coding sequence to increase the amount ofthe mature message that accumulates in the cytosol. Inclusion of aspliceable intron in the transcription unit in both plant and animalexpression constructs has been shown to increase gene expression at boththe mRNA and protein levels up to 1000-fold. Buchman and Berg, (1988)Mol. Cell Biol. 8:4395-4405; Callis, et al., (1987) Genes Dev.1:1183-1200. Such intron enhancement of gene expression is typicallygreatest when placed near the 5′ end of the transcription unit. Use ofmaize introns Adhl-S intron1, 2 and 6, the Bronze-1 intron are known inthe art. See generally, The Maize Handbook, Chapter 116, Freeling andWalbot, Eds., Springer, New York (1994).

The vector comprising the sequences from a polynucleotide of the presentinvention will typically comprise a marker gene which confers aselectable phenotype on plant cells. Usually, the selectable marker genewill encode antibiotic resistance, with suitable genes including genescoding for resistance to the antibiotic spectinomycin (e.g., the aadagene), the streptomycin phosphotransferase (SPT) gene coding forstreptomycin resistance, the neomycin phosphotransferase (NPTII) geneencoding kanamycin or genetic in resistance, the hygromycinphosphotransferase (HPT) gene coding for hygromycin resistance, genescoding for resistance to herbicides which act to inhibit the action ofacetolactate synthase (ALS), in particular the sulfonylurea-typeherbicides (e.g., the acetolactate synthase (ALS) gene containingmutations leading to such resistance in particular the S4 and/or Hramutations), genes coding for resistance to herbicides which act toinhibit action of glutamine synthase, such as phosphinothricin or basta(e.g., the bar gene) or other such genes known in the art. The bar geneencodes resistance to the herbicide basta, the nptII gene encodesresistance to the antibiotic kanamycin, and the ALS gene encodesresistance to the herbicide chlorsulfuron.

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of Agrobacterium tumefaciens described by Rogers, et al.,(1987) Meth. in Enzymol. 153:253-277. These vectors are plantintegrating vectors in that on transformation, the vectors integrate aportion of vector DNA into the genome of the host plant. Exemplary A.tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 ofSchardl, et al., (1987) Gene 61:1-11 and Berger, et al., (1989) Proc.Natl. Acad. Sci. USA 86:8402-8406. Another useful vector herein isplasmid pBI101.2 that is available from Clontech Laboratories, Inc.(Palo Alto, Calif.).

A polynucleotide of the present invention can be expressed in eithersense or antisense orientation as desired. It will be appreciated thatcontrol of gene expression in either sense or anti-sense orientation canhave a direct impact on the observable plant characteristics. Antisensetechnology can be conveniently used to inhibit gene expression inplants. To accomplish this, a nucleic acid segment from the desired geneis cloned and operably linked to a promoter such that the anti-sensestrand of RNA will be transcribed. The construct is then transformedinto plants and the antisense strand of RNA is produced.

In plant cells, it has been shown that antisense RNA inhibits geneexpression by preventing the accumulation of mRNA which encodes theenzyme of interest, see, e.g., Sheehy, et al., (1988) Proc. Nat'l. Acad.Sci. (USA) 85:8805-8809 and Hiatt, et al., U.S. Pat. No. 4,801,340.

Another method of suppression is sense suppression. Introduction ofnucleic acid configured in the sense orientation has been shown to be aneffective means by which to block the transcription of target genes. Foran example of the use of this method to modulate expression ofendogenous genes see, Napoli, et al., (1990) The Plant Cell 2:279-289and U.S. Pat. No. 5,034,323.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA cleaving activity upon them,thereby increasing the activity of the constructs. The design and use oftarget RNA-specific ribozymes is described in Haseloff, et al., (1988)Nature 334:585-591. A variety of cross-linking agents, alkylating agentsand radical generating species as pendant groups on polynucleotides ofthe present invention can be used to bind, label, detect, and/or cleavenucleic acids. For example, Vlassov, et al., (1986) Nucleic Acids Res14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre, et al., (1985) Biochimie 67:785 789. Iverson and Dervan.

The present invention further provides a protein comprising apolypeptide having a specified sequence identity with a polypeptide ofthe present invention. The percentage of sequence identity is an integerselected from the group consisting of from 60 to 99. Exemplary sequenceidentity values include 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% and 99% to a full-length sequence of theinvention.

As those of skill will appreciate, the present invention includescatalytically active polypeptides of the present invention (i.e.,enzymes). Catalytically active polypeptides have a specific activity ofat least 20%, 30% or 40%, and preferably at least 50%, 60% or 70% andmost preferably at least 80%, 90% or 95% that of the native(non-synthetic), endogenous polypeptide. Further, the substratespecificity (kcat/Km) is optionally substantially similar to the native(non-synthetic), endogenous polypeptide. Typically, the Km will be atleast 30%, 40% or 50%, that of the native (non-synthetic), endogenouspolypeptide and more preferably at least 60%, 70%, 80% or 90%. Methodsof assaying and quantifying measures of enzymatic activity and substratespecificity (heat/Km), are well known to those of skill in the art.

Generally, the proteins of the present invention will, when presented asan immunogen, elicit production of an antibody specifically reactive toa polypeptide of the present invention. Further, the proteins of thepresent invention will not bind to antisera raised against a polypeptideof the present invention which has been fully immunosorbed with the samepolypeptide. Immunoassays for determining binding are well known tothose of skill in the art. A preferred immunoassay is a competitiveimmunoassay as discussed, infra. Thus, the proteins of the presentinvention can be employed as immunogens for constructing antibodiesimmunoreactive to a protein of the present invention for such exemplaryutilities as immunoassays or protein purification techniques.

Expression of Proteins in Host Cells

Using the nucleic acids of the present invention, one may express aprotein of the present invention in a recombinantly engineered cell suchas bacteria, yeast, insect, mammalian or preferably plant cells. Thecells produce the protein in a non-natural condition (e.g., in quantity,composition, location and/or time), because they have been geneticallyaltered through human intervention to do so.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of a nucleic acidencoding a protein of the present invention. No attempt to describe indetail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

In brief summary, the expression of isolated nucleic acids encoding aprotein of the present invention will typically be achieved by operablylinking, for example, the DNA or cDNA to a promoter (which is eitherconstitutive or regulatable), followed by incorporation into anexpression vector. The vectors can be suitable for replication andintegration in either prokaryotes or eukaryotes. Typical expressionvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of theDNA encoding a protein of the present invention. To obtain high levelexpression of a cloned gene, it is desirable to construct expressionvectors which contain, at the minimum, a strong promoter to directtranscription, a ribosome binding site for translational initiation anda transcription/translation terminator. One of skill would recognizethat modifications can be made to a protein of the present inventionwithout diminishing its biological activity. Some modifications may bemade to facilitate the cloning, expression, or incorporation of thetargeting molecule into a fusion protein.

Such modifications are well known to those of skill in the art andinclude, for example, a methionine added at the amino terminus toprovide an initiation site, or additional amino acids (e.g., poly His)placed on either terminus to create conveniently located purificationsequences. Restriction sites or termination codons can also beintroduced.

A. Expression in Prokaryotes

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta lactamase (penicillinase) and lactose (lac) promoter systems(Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promotersystem (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057) and thelambda derived P L promoter and N-gene ribosome binding site (Shimatake,et al., (1981) Nature 292:128). The inclusion of selection markers inDNA vectors transfected in E. coli is also useful. Examples of suchmarkers include genes specifying resistance to ampicillin, tetracycline,or chloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva, et al., (1983) Gene22:229-235; Mosbach, et al., (1983) Nature 302:543-545).

B. Expression in Eukaryotes

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, a polynucleotide of the presentinvention can be expressed in these eukaryotic systems. In someembodiments, transformed/transfected plant cells, as discussed infra,are employed as expression systems for production of the proteins of theinstant invention.

Synthesis of heterologous proteins in yeast is well known. Sherman, etal., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) isa well recognized work describing the various methods available toproduce the protein in yeast. Two widely utilized yeast for productionof eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.Vectors, strains, and protocols for expression in Saccharomyces andPichia are known in the art and available from commercial suppliers(e.g., Invitrogen).

Suitable vectors usually have expression control sequences, such aspromoters, including 3-phosphoglycerate kinase or alcohol oxidase and anorigin of replication, termination sequences and the like as desired.

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysate. The monitoring of the purification process canbe accomplished by using Western blot techniques or radioimmunoassay orother standard immunoassay techniques.

The sequences encoding proteins of the present invention can also beligated to various expression vectors for use in transfecting cellcultures of, for instance, mammalian, insect or plant origin.Illustrative of cell cultures useful for the production of the peptidesare mammalian cells. Mammalian cell systems often will be in the form ofmonolayers of cells although mammalian cell suspensions may also beused. A number of suitable host cell lines capable of expressing intactproteins have been developed in the art, and include the HEK293, BHK21and CHO cell lines. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter (e.g., the CMV promoter, a HSVtk promoter or pgk(phosphoglycerate kinase) promoter), an enhancer (Queen, et al., (1986)Immunol. Rev. 89:49) and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site) and transcriptional terminatorsequences. Other animal cells useful for production of proteins of thepresent invention are available, for instance, from the American TypeCulture Collection.

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, army worm, moth andDrosophila cell lines such as a Schneider cell line (see, Schneider,(1987) Embryol. Exp. Morphol. 27:353-365.

As with yeast, when higher animal or plant host cells are employed,polyadenylation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenylation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague, et al.,(1983) J. Virol. 45:773-781). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors. Saveria-Campo,Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA CloningVol. II a Practical Approach, Glover, Ed., IRL Press, Arlington, Va. pp.213-238 (1985).

Increasing the Activity and/or Level of an ETO1 Polypeptide

Methods are provided to increase the activity and/or level of the ETO1polypeptide of the invention. An increase in the level and/or activityof the ETO1 polypeptide of the invention can be achieved by providing tothe plant an ETO1 polypeptide. The ETO1 polypeptide can be provided byintroducing the amino acid sequence encoding the ETO1 polypeptide intothe plant, introducing into the plant a nucleotide sequence encoding anETO1 polypeptide or alternatively by modifying a genomic locus encodingthe ETO1 polypeptide of the invention.

As discussed elsewhere herein, many methods are known in the art forproviding a polypeptide to a plant including, but not limited to, directintroduction of the polypeptide into the plant, introducing into theplant (transiently or stably) a polynucleotide construct encoding apolypeptide having enhanced activity. It is also recognized that themethods of the invention may employ a polynucleotide that is not capableof directing, in the transformed plant, the expression of a protein oran RNA. Thus, the level and/or activity of an ETO1 polypeptide may beincreased by altering the gene encoding the ETO1 polypeptide or itspromoter. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al.,PCT Application Serial Number PCT/US93/03868. Therefore mutagenizedplants that carry mutations in ETO1 genes, where the mutations increaseexpression of the ETO1 or increase the activity of the encoded ETO1polypeptide, are provided.

Reducing the Activity and/or Level of an ETO1 Polypeptide

In certain embodiments, methods are provided to reduce or eliminate theactivity of an ETO1 polypeptide of the invention by transforming a plantcell with an expression cassette that expresses a polynucleotide thatinhibits the expression of the ETO1 polypeptide. The polynucleotide mayinhibit the expression of the ETO1 polypeptide directly, by preventingtranscription or translation of the ETO1 associated messenger RNA orindirectly, by encoding a polypeptide that inhibits the transcription ortranslation of an ETO1 gene encoding ETO1 polypeptide. Methods forinhibiting or eliminating the expression of a gene in a plant are wellknown in the art and any such method may be used in the presentinvention to inhibit the expression of ETO1 polypeptide.

In accordance with the present invention, the expression of an ETO1polypeptide is inhibited if the protein level of the ETO1 polypeptide isless than 70% of the protein level of the same ETO1 polypeptide in aplant that has not been genetically modified or mutagenized to inhibitthe expression of that ETO1 polypeptide. In particular embodiments ofthe invention, the protein level of the ETO1 polypeptide in a modifiedplant according to the invention is less than 60%, less than 50%, lessthan 40%, less than 30%, less than 20%, less than 10%, less than 5% orless than 2% of the protein level of the same ETO1 polypeptide in aplant that is not a mutant or that has not been genetically modified toinhibit the expression of that ETO1 polypeptide. The expression level ofthe ETO1 polypeptide may be measured directly, for example, by assayingfor the level of ETO1 polypeptide expressed in the plant cell or plant,or indirectly, for example, by measuring the ethylene response in theplant cell or plant, or by measuring the phenotypic changes in theplant. Methods for performing such assays are described elsewhereherein.

In other embodiments of the invention, the activity of the ETO1polypeptide is reduced or eliminated by transforming a plant cell withan expression cassette comprising a polynucleotide encoding apolypeptide that inhibits the activity of an ETO1 polypeptide. Theactivity of an ETO1 polypeptide is inhibited according to the presentinvention if the activity of the ETO1 polypeptide is less than 70% ofthe activity of the same ETO1 polypeptide in a plant that has not beenmodified to inhibit the activity of that polypeptide. In particularembodiments of the invention, the activity of the ETO1 polypeptide in amodified plant according to the invention is less than 60%, less than50%, less than 40%, less than 30%, less than 20%, less than 10% or lessthan 5% of the activity of the same polypeptide in a plant that that hasnot been modified to inhibit the expression of that ETO1 polypeptide.The activity of an ETO1 polypeptide is “eliminated” according to theinvention when it is not detectable by the assay methods describedelsewhere herein. Methods of determining the alteration of activity ofan ETO1 polypeptide are described elsewhere herein.

In other embodiments, the activity of an ETO1 polypeptide may be reducedor eliminated by disrupting the gene encoding the ETO1 polypeptide. Theinvention encompasses mutagenized plants that carry mutations in ETO1genes, where the mutations reduce expression of the associated gene orinhibit the activity of the encoded ETO1 polypeptide.

Thus, many methods may be used to reduce or eliminate the activity of anETO1 polypeptide. In addition, more than one method may be used toreduce the activity of a single ETO1 polypeptide.

1. Polynucleotide-Based Methods:

In some embodiments of the present invention, a plant is transformedwith an expression cassette that is capable of expressing apolynucleotide that inhibits the expression of an ETO1 polypeptide ofthe invention. The term “expression” as used herein refers to thebiosynthesis of a gene product, including the transcription and/ortranslation of said gene product. For example, for the purposes of thepresent invention, an expression cassette capable of expressing apolynucleotide that inhibits the expression of at least one ETO1polypeptide is an expression cassette capable of producing an RNAmolecule that inhibits the transcription and/or translation of at leastone ETO1 polypeptide of the invention. The “expression” or “production”of a protein or polypeptide from a DNA molecule refers to thetranscription and translation of the coding sequence to produce theprotein or polypeptide, while the “expression” or “production” of aprotein or polypeptide from an RNA molecule refers to the translation ofthe RNA coding sequence to produce the protein or polypeptide.

Examples of polynucleotides and methodology that inhibit the expressionof an ETO1 polypeptide include, sense suppression, cosuppression,antisense suppression, double stranded RNA interference, hairpin RNAInterference, intron-containing hairpin RNA interference,amplicon-mediated interference, ribozymes, small interfering RNA ormicro RNA. Other methods of inhibition can include polypeptide-basedinhibition of gene expression, or of protein activity as well as genedisruption.

2. Mutant Plants with Reduced Activity:

Additional methods for decreasing or eliminating the expression ofendogenous genes in plants are also known in the art and can besimilarly applied to the instant invention. These methods include otherforms of mutagenesis, such as ethyl methanesulfonate-inducedmutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesisused in a reverse genetics sense (with PCR) to identify plant lines inwhich the endogenous gene has been deleted. For examples of thesemethods see, Ohshima, et al., (1998) Virology 243:472-481; Okubara, etal., (1994) Genetics 137:867-874 and Quesada, et al., (2000) Genetics154:421-436; each of which is herein incorporated by reference. Inaddition, a fast and automatable method for screening for chemicallyinduced mutations, TILLING (Targeting Induced Local Lesions In Genomes),using denaturing HPLC or selective endonuclease digestion of selectedPCR products is also applicable to the instant invention. See, McCallum,et al., (2000) Nat. Biotechnol. 18:455-457, herein incorporated byreference.

Mutations that impact gene expression or that interfere with thefunction (enhanced activity) of the encoded protein are well known inthe art. Insertional mutations in gene exons usually result innull-mutants. Mutations in conserved residues are particularly effectivein inhibiting the activity of the encoded protein. Conserved residues ofplant ETO1 polypeptides suitable for mutagenesis with the goal toeliminate activity have been described. Such mutants can be isolatedaccording to well-known procedures, and mutations in different ETO1associated loci can be stacked by genetic crossing. See, for example,Gruis, et al., (2002) Plant Cell 14:2863-2882.

The invention encompasses additional methods for reducing or eliminatingthe activity of one or more ETO1 polypeptide. Examples of other methodsfor altering or mutating a genomic nucleotide sequence in a plant areknown in the art and include, but are not limited to, the use of RNA:DNAvectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides and recombinogenic oligonucleobases. Such vectors andmethods of use are known in the art. See, for example, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984;each of which are herein incorporated by reference. See also, WO1998/49350, WO 1999/07865, WO 1999/25821 and Beetham, et al., (1999)Proc. Natl. Acad. Sci. USA 96:8774-8778, each of which is hereinincorporated by reference.

Transfection/Transformation of Cells

The method of transformation/transfection is not critical to the instantinvention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be directly applied.

Accordingly, a wide variety of methods have been developed to insert aDNA sequence into the genome of a host cell to obtain the transcriptionand/or translation of the sequence to effect phenotypic changes in theorganism. Thus, any method which provides for effectivetransformation/transfection may be employed.

A. Plant Transformation

A DNA sequence coding for the desired polypeptide of the presentinvention, for example a cDNA or a genomic sequence encoding a fulllength protein, will be used to construct a recombinant expressioncassette which can be introduced into the desired plant.

Isolated nucleic acid acids of the present invention can be introducedinto plants according to techniques known in the art. Generally,recombinant expression cassettes as described above and suitable fortransformation of plant cells are prepared. Techniques for transforminga wide variety of higher plant species are well known and described inthe technical, scientific and patent literature. See, for example,Weising et al., (1988) Ann. Rev. Genet. 22:421-477. For example, the DNAconstruct may be introduced directly into the genomic DNA of the plantcell using techniques such as electroporation, polyethylene glycol(PEG), poration, particle bombardment, silicon fiber delivery ormicroinjection of plant cell protoplasts or embryogenic callus. See,e.g., Tomes, et al., Direct DNA Transfer into Intact Plant Cells ViaMicroprojectile Bombardment. pp. 197213 in Plant Cell, Tissue and OrganCulture, Fundamental Methods. eds. Gamborg and Phillips. Springer-VerlagBerlin Heidelberg New York, 1995. Alternatively, the DNA constructs maybe combined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. See, U.S. Pat. No. 5,591,616.

The introduction of DNA constructs using PEG precipitation is describedin Paszkowski, et al., (1984) Embo J. 3:2717-2722. Electroporationtechniques are described in Fromm, et al., (1985) Proc. Natl. Acad. Sci.(USA) 82:5824. Ballistic transformation techniques are described inKlein et al., (1987) Nature 327:70-73.

Agrobacterium tumefaciens-mediated transformation techniques are welldescribed in the scientific literature. See, for example Horsch, et al.,(1984) Science 233:496-498 and Fraley et al., (1983) Proc. Natl. Acad.Sci. (USA) 80:4803. Although Agrobacterium is useful primarily indicots, certain monocots can be transformed by Agrobacterium. Forinstance, Agrobacterium transformation of maize is described in U.S.Pat. No. 5,550,318.

Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein and Fuller In: Genetic Engineering, vol. 6, P W J Rigby,Ed., London, Academic Press, 1987; and Lichtenstein and Draper, In: DNACloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press, 1985), PCTApplication Number PCT/US87/02512 (WO 1988/02405 published Apr. 7, 1988)describes the use of A. rhizogenes strain A4 and its Ri plasmid alongwith A. tumefaciens vectors pARC8 orpARC16 (2) liposome-mediated DNAuptake (see, e.g., Freeman, et al., (1984) Plant Cell Physiol. 25:1353),(3) the vortexing method (see, e.g., Kindle, (1990) Proc. Natl. Acad.Sci., (USA) 87:1228).

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou, et al., (1983) Methods in Enzymology101:433; Hess, (1987) Intern Rev. Cytol. 107:367; Luo, et al., (1988)Plant Mol. Biol. Reporter 6:165. Expression of polypeptide coding genescan be obtained by injection of the DNA into reproductive organs of aplant as described by Pena, et al., (1987) Nature 325:274.

DNA can also be injected directly into the cells of immature embryos andthe rehydration of desiccated embryos as described by Neuhaus, et al.,(1987) Theor. Appl. Genet. 75:30 and Benbrook, et al., in ProceedingsBio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986). A varietyof plant viruses that can be employed as vectors are known in the artand include cauliflower mosaic virus (CaMV), geminivirus, brome mosaicvirus, and tobacco mosaic virus.

B. Transfection of Prokaryotes, Lower Eukaryotes, and Animal Cells

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextran, electroporation,biolistics and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art. Kuchler,Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson andRoss, Inc. (1977).

Synthesis of Proteins

The proteins of the present invention can be constructed usingnon-cellular synthetic methods. Solid phase synthesis of proteins ofless than about 50 amino acids in length may be accomplished byattaching the C-terminal amino acid of the sequence to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology, Vol. 2: Special Methods inPeptide Synthesis, Part A.; Merrifield, et al., (1963) J. Am. Chem. Soc.85:2149-2156 and Stewart, et al., Solid Phase Peptide Synthesis, 2nded., Pierce Chem. Co., Rockford, III. (1984). Proteins of greater lengthmay be synthesized by condensation of the amino and carboxy termini ofshorter fragments. Methods of forming peptide bonds by activation of acarboxy terminal end (e.g., by the use of the coupling reagentN,N′-dicycylohexylcarbodiimide) are known to those of skill.

Purification of Proteins

The proteins of the present invention may be purified by standardtechniques well known to those of skill in the art. Recombinantlyproduced proteins of the present invention can be directly expressed orexpressed as a fusion protein. The recombinant protein is purified by acombination of cell lysis (e.g., sonication, French press) and affinitychromatography. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredrecombinant protein.

The proteins of this invention, recombinant or synthetic, may bepurified to substantial purity by standard techniques well known in theart, including detergent solubilization, selective precipitation withsuch substances as ammonium sulfate, column chromatography,immunopurification methods and others. See, for instance, Scopes,Protein Purification: Principles and Practice, Springer-Verlag: New York(1982); Deutscher, Guide to Protein Purification, Academic Press (1990).For example, antibodies may be raised to the proteins as describedherein. Purification from E. coli can be achieved following proceduresdescribed in U.S. Pat. No. 4,511,503. The protein may then be isolatedfrom cells expressing the protein and further purified by standardprotein chemistry techniques as described herein. Detection of theexpressed protein is achieved by methods known in the art and include,for example, radioimmunoassays, Western blotting techniques orimmunoprecipitation.

Transgenic Plant Regeneration

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium. For transformation and regeneration of maize see,Gordon-Kamm, et al., (1990) The Plant Cell 2:603-618.

Plants cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans, et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmillan Publishing Company, New York, pp. 124-176 (1983) and Binding,Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp.21-73 (1985).

The regeneration of plants containing the foreign gene introduced byAgrobacterium from leaf explants can be achieved as described by Horsch,et al., (1985) Science 227:1229-1231. In this procedure, transformantsare grown in the presence of a selection agent and in a medium thatinduces the regeneration of shoots in the plant species beingtransformed as described by Fraley, et al., (1983) Proc. Natl. Acad.Sci. USA 80:4803. This procedure typically produces shoots within two tofour weeks and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. Transgenic plants of the presentinvention may be fertile or sterile.

Regeneration can also be obtained from plant callus, explants, organs,or parts thereof. Such regeneration techniques are described generallyin Kleen, et al., (1987) Ann. Rev. of Plant Phys. 38:467-486. Theregeneration of plants from either single plant protoplasts or variousexplants is well known in the art. See, for example, Methods for PlantMolecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press,Inc., San Diego, Calif. (1988). This regeneration and growth processincludes the steps of selection of transformant cells and shoots,rooting the transformant shoots and growth of the plantlets in soil. Formaize cell culture and regeneration see generally, The Maize Handbook,Freeling and Walbot, Eds., Springer, New York (1994); Corn and CornImprovement, 3rd edition, Sprague and Dudley Eds., American Society ofAgronomy, Madison, Wis. (1988).

One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. In vegetatively propagated crops, maturetransgenic plants can be propagated by the taking of cuttings or bytissue culture techniques to produce multiple identical plants.

Selection of desirable transgenics is made and new varieties areobtained and propagated vegetatively for commercial use. In seedpropagated crops, mature transgenic plants can be self crossed toproduce a homozygous inbred plant. The inbred plant produces seedcontaining the newly introduced heterologous nucleic acid. These seedscan be grown to produce plants that would produce the selectedphenotype.

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are included in the invention,provided that these parts comprise cells comprising the isolated nucleicacid of the present invention. Progeny and variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced nucleic acidsequences. Transgenic plants expressing the selectable marker can bescreened for transmission of the nucleic acid of the present inventionby, for example, standard immunoblot and DNA detection techniques.Transgenic lines are also typically evaluated on levels of expression ofthe heterologous nucleic acid. Expression at the RNA level can bedetermined initially to identify and quantitate expression-positiveplants. Standard techniques for RNA analysis can be employed and includePCR amplification assays using oligonucleotide primers designed toamplify only the heterologous RNA templates and solution hybridizationassays using heterologous nucleic acid-specific probes. The RNA-positiveplants can then analyzed for protein expression by Western immunoblotanalysis using the specifically reactive antibodies of the presentinvention. In addition, in situ hybridization and immunocytochemistryaccording to standard protocols can be done using heterologous nucleicacid specific polynucleotide probes and antibodies, respectively, tolocalize sites of expression within transgenic tissue. Generally, anumber of transgenic lines are usually screened for the incorporatednucleic acid to identify and select plants with the most appropriateexpression profiles.

A preferred embodiment is a transgenic plant that is homozygous for theadded heterologous nucleic acid; i.e., a transgenic plant that containstwo added nucleic acid sequences, one gene at the same locus on eachchromosome of a chromosome pair. A homozygous transgenic plant can beobtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered expression of a polynucleotide of the present invention relativeto a control plant (i.e., native, nontransgenic). Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated.

Modulation of Polypeptide Levels and/or Composition

The present invention further provides a method for modulating (i.e.,increasing or decreasing) the concentration or ratio of the polypeptidesof the present invention in a plant or part thereof. Modulation can beeffected by increasing or decreasing the concentration and/or the ratioof the polypeptides of the present invention in a plant.

The method comprises introducing into a plant cell a recombinantexpression cassette comprising a polynucleotide of the present inventionas described above to obtain a transformed plant cell, culturing thetransformed plant cell under plant cell growing conditions and inducingor repressing expression of a polynucleotide of the present invention inthe plant for a time sufficient to modulate concentration and/or theratios of the polypeptides in the plant or plant part.

In some embodiments, the concentration and/or ratios of polypeptides ofthe present invention in a plant may be modulated by altering, in vivoor in vitro, the promoter of a gene to up- or down-regulate geneexpression. In some embodiments, the coding regions of native genes ofthe present invention can be altered via substitution, addition,insertion, or deletion to decrease activity of the encoded enzyme. See,e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT PatentApplication Number PCT/US93/03868. And in some embodiments, an isolatednucleic acid (e.g., a vector) comprising a promoter sequence istransfected into a plant cell.

Subsequently, a plant cell comprising the promoter operably linked to apolynucleotide of the present invention is selected for by means knownto those of skill in the art, such as, but not limited to, Southernblot, DNA sequencing or PCR analysis using primers specific to thepromoter and to the gene and detecting amplicons produced therefrom. Aplant or plant part altered or modified by the foregoing embodiments isgrown under plant forming conditions for a time sufficient to modulatethe concentration and/or ratios of polypeptides of the present inventionin the plant. Plant forming conditions are well known in the art anddiscussed briefly, supra.

In general, concentration or the ratios of the polypeptides is increasedor decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or90% relative to a native control plant, plant part, or cell lacking theaforementioned recombinant expression cassette. Modulation in thepresent invention may occur during and/or subsequent to growth of theplant to the desired stage of development. Modulating nucleic acidexpression temporally and/or in particular tissues can be controlled byemploying the appropriate promoter operably linked to a polynucleotideof the present invention in, for example, sense or antisense orientationas discussed in greater detail, supra. Induction of expression of apolynucleotide of the present invention can also be controlled byexogenous administration of an effective amount of inducing compound.Inducible promoters and inducing compounds which activate expressionfrom these promoters are well known in the art. In preferredembodiments, the polypeptides of the present invention are modulated inmonocots, particularly maize.

Molecular Markers

The present invention provides a method of genotyping a plant comprisinga polynucleotide of the present invention. Optionally, the plant is amonocot, such as maize or sorghum. Genotyping provides a means ofdistinguishing homologs of a chromosome pair and can be used todifferentiate segregants in a plant population. Molecular marker methodscan be used for phylogenetic studies, characterizing geneticrelationships among crop varieties, identifying crosses or somatichybrids, localizing chromosomal segments affecting monogenic traits, mapbased cloning and the study of quantitative inheritance. See, e.g.,Clark, Ed., Plant Molecular Biology: A Laboratory Manual. Berlin,Springer Verlag, 1997, Chapter 7. For molecular marker methods, seegenerally, “The DNA Revolution” in: Paterson, Genome Mapping in Plants(Austin, Tex., Academic Press/R. G. Landis Company, 1996) pp. 7-21.

The particular method of genotyping in the present invention may employany number of molecular marker analytic techniques such as, but notlimited to, restriction fragment length polymorphisms (RFLPs). RFLPs arethe product of allelic differences between DNA restriction fragmentsresulting from nucleotide sequence variability. As is well known tothose of skill in the art, RFLPs are typically detected by extraction ofgenomic DNA and digestion with a restriction enzyme. Generally, theresulting fragments are separated according to size and hybridized witha probe; single copy probes are preferred. Restriction fragments fromhomologous chromosomes are revealed.

Differences in fragment size among alleles represent an RFLP. Thus, thepresent invention further provides a means to follow segregation of agene or nucleic acid of the present invention as well as chromosomalsequences genetically linked to these genes or nucleic acids using suchtechniques as RFLP analysis. Linked chromosomal sequences are within 50centiMorgans (cM), often within 40 or 30 cM, preferably within 20 or 10cM, more preferably within 5, 3, 2 or 1 cM of a gene of the presentinvention.

In the present invention, the nucleic acid probes employed for molecularmarker mapping of plant nuclear genomes selectively hybridize, underselective hybridization conditions, to a gene encoding a polynucleotideof the present invention. In preferred embodiments, the probes areselected from polynucleotides of the present invention.

Typically, these probes are cDNA probes or restriction-enzyme treated(e.g., Pst I) genomic clones. The length of the probes is discussed ingreater detail, supra, but are typically at least 15 bases in length,more preferably at least 20, 25, 30, 35, 40 or 50 bases in length.Generally, however, the probes are less than about 1 kilobase in length.Preferably, the probes are single copy probes that hybridize to a uniquelocus in a haploid chromosome complement. Some exemplary restrictionenzymes employed in RFLP mapping are EcoRl, EcoRv and Sstl. As usedherein the term “restriction enzyme” includes reference to a compositionthat recognizes and alone or in conjunction with another composition,cleaves at a specific nucleotide sequence.

The method of detecting an RFLP comprises the steps of (a) digestinggenomic DNA of a plant with a restriction enzyme; (b) hybridizing anucleic acid probe, under selective hybridization conditions, to asequence of a polynucleotide of the present of said genomic DNA; (c)detecting therefrom a RFLP. Other methods of differentiating polymorphic(allelic) variants of polynucleotides of the present invention can behad by utilizing molecular marker techniques well known to those ofskill in the art including such techniques as: 1) single strandedconformation analysis (SSCA); 2) denaturing gradient gel electrophoresis(DGGE); 3) RNase protection assays; 4) allele-specific oligonucleotides(ASOs); 5) the use of proteins which recognize nucleotide mismatches,such as the E. coli mutS protein and 6) allele-specific PCR. Otherapproaches based on the detection of mismatches between the twocomplementary DNA strands include clamped denaturing gel electrophoresis(CDGE); heteroduplex analysis (HA); and chemical mismatch cleavage(CMC). Thus, the present invention further provides a method ofgenotyping comprising the steps of contacting, under stringenthybridization conditions, a sample suspected of comprising apolynucleotide of the present invention with a nucleic acid probe.Generally, the sample is a plant sample; preferably, a sample suspectedof comprising a maize polynucleotide of the present invention (e.g.,gene, mRNA). The nucleic acid probe selectively hybridizes, understringent conditions, to a subsequence of a polynucleotide of thepresent invention comprising a polymorphic marker. Selectivehybridization of the nucleic acid probe to the polymorphic markernucleic acid sequence yields a hybridization complex. Detection of thehybridization complex indicates the presence of that polymorphic markerin the sample. In preferred embodiments, the nucleic acid probecomprises a polynucleotide of the present invention.

UTRs and Codon Preference

In general, translational efficiency has been found to be regulated byspecific sequence elements in the 5′ non-coding or untranslated region(5′ UTR) of the RNA. Positive sequence motifs include translationalinitiation consensus sequences (Kozak, (1987) Nucleic Acids Res.15:8125) and the 7-methylguanosine cap structure (Drummond, et al.,(1985) Nucleic Acids Res. 13:7375). Negative elements include stableintramolecular 5′ UTR stem-loop structures (Muesing, et al., (1987) Cell48:691) and AUG sequences or short open reading frames preceded by anappropriate AUG in the 5′ UTR (Kozak, supra, Rao, et al., (1988) Mol.and Cell. Biol. 8:284). Accordingly, the present invention provides 5′and/or 3′ untranslated regions for modulation of translation ofheterologous coding sequences.

Further, the polypeptide-encoding segments of the polynucleotides of thepresent invention can be modified to alter codon usage. Altered codonusage can be employed to alter translational efficiency and/or tooptimize the coding sequence for expression in a desired host such as tooptimize the codon usage in a heterologous sequence for expression inmaize. Codon usage in the coding regions of the polynucleotides of thepresent invention can be analyzed statistically using commerciallyavailable software packages such as “Codon Preference” available fromthe University of Wisconsin Genetics Computer Group (see, Devereaux, etal., (1984) Nucleic Acids Res. 12:387-395) or MacVector 4.1 (EastmanKodak Co., New Haven, Conn.). Thus, the present invention provides acodon usage frequency characteristic of the coding region of at leastone of the polynucleotides of the present invention. The number ofpolynucleotides that can be used to determine a codon usage frequencycan be any integer from 1 to the number of polynucleotides of thepresent invention as provided herein. Optionally, the polynucleotideswill be full-length sequences. An exemplary number of sequences forstatistical analysis can be at least 1, 5, 10, 20, 50 or 100.

Sequence Shuffling

The present invention provides methods for sequence shuffling usingpolynucleotides of the present invention, and compositions resultingtherefrom. Sequence shuffling is described in PCT ApplicationPublication Number WO 1997/20078. See also, Zhang, et al., (1997) Proc.Natl. Acad. Sci. USA 94:4504-4509. Generally, sequence shufflingprovides a means for generating libraries of polynucleotides having adesired characteristic which can be selected or screened for. Librariesof recombinant polynucleotides are generated from a population ofrelated sequence polynucleotides which comprise sequence regions whichhave substantial sequence identity and can be homologously recombined invitro or in vivo. The population of sequence-recombined polynucleotidescomprises a subpopulation of polynucleotides which possess desired oradvantageous characteristics and which can be selected by a suitableselection or screening method. The characteristics can be any propertyor attribute capable of being selected for or detected in a screeningsystem, and may include properties of: an encoded protein, atranscriptional element, a sequence controlling transcription, RNAprocessing, RNA stability, chromatin conformation, translation or otherexpression property of a gene or transgene, a replicative element, aprotein-binding element, or the like, such as any feature which confersa selectable or detectable property. In some embodiments, the selectedcharacteristic will be a decreased Km and/or increased KCat over thewild-type protein as provided herein. In other embodiments, a protein orpolynucleotide generated from sequence shuffling will have a ligandbinding affinity greater than the non-shuffled wild-type polynucleotide.The increase in such properties can be at least 110%, 120%, 130%, 140%or at least 150% of the wild-type value.

Generic and Consensus Sequences

Polynucleotides and polypeptides of the present invention furtherinclude those having: (a) a generic sequence of at least two homologouspolynucleotides or polypeptides, respectively, of the present inventionand (b) a consensus sequence of at least three homologouspolynucleotides or polypeptides, respectively, of the present invention.The generic sequence of the present invention comprises each species ofpolypeptide or polynucleotide embraced by the generic polypeptide orpolynucleotide sequence, respectively. The individual speciesencompassed by a polynucleotide having an amino acid or nucleic acidconsensus sequence can be used to generate antibodies or produce nucleicacid probes or primers to screen for homologs in other species, genera,families, orders, classes, phyla, or kingdoms. For example, apolynucleotide having a consensus sequence from a gene family of Zeamays can be used to generate antibody or nucleic acid probes or primersto other Gramineae species such as wheat, rice, or sorghum.

Alternatively, a polynucleotide having a consensus sequence generatedfrom orthologous genes can be used to identify or isolate orthologs ofother taxa. Typically, a polynucleotide having a consensus sequence willbe at least 25, 30, or 40 amino acids in length, or 20, 30, 40, 50, 100or 150 nucleotides in length. As those of skill in the art are aware, aconservative amino acid substitution can be used for amino acids whichdiffer amongst aligned sequence but are from the same conservativesubstitution group as discussed above. Optionally, no more than 1 or 2conservative amino acids are substituted for each 10 amino acid lengthof consensus sequence.

Similar sequences used for generation of a consensus or generic sequenceinclude any number and combination of allelic variants of the same gene,orthologous or paralogous sequences as provided herein. Optionally,similar sequences used in generating a consensus or generic sequence areidentified using the BLAST algorithm's smallest sum probability (P(N)).Various suppliers of sequence-analysis software are listed in chapter 7of Current Protocols in Molecular Biology, Ausubel, et al., Eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc. (Supplement 30).

A polynucleotide sequence is considered similar to a reference sequenceif the smallest sum probability in a comparison of the test nucleic acidto the reference nucleic acid is less than about 0.1, more preferablyless than about 0.01, or 0.001 and most preferably less than about0.0001 or 0.00001. Similar polynucleotides can be aligned and aconsensus or generic sequence generated using multiple sequencealignment software available from a number of commercial suppliers, suchas the Genetics Computer Group's (Madison, Wis.) PILEUP software, VectorNTI's (North Bethesda, Md.) ALIGNX or Genecode's (Ann Arbor, Mich.)SEQUENCHER. Conveniently, default parameters of such software can beused to generate consensus or generic sequences.

Machine Applications

The present invention provides machines, articles of manufacture, andprocesses for identifying, modeling or analyzing the polynucleotides andpolypeptides of the present invention. Identification methods permitidentification of homologues of the polynucleotides or polypeptides ofthe present invention while modeling and analysis methods permitrecognition of structural or functional features of interest.

A. Machines: Data Processing Systems

In one embodiment, the present invention provides a machine having: 1) amemory comprising data representing at least one genetic sequence, 2) agenetic identification, analysis, or modeling program with access to thedata, 3) a data processor which executes instructions according to theprogram using the genetic sequence or a subsequence thereof and 4) anoutput for storing or displaying the results of the data processing.

The machine of the present invention is a data processing system,typically a digital computer. The term “computer” includes one orseveral desktop or portable computers, computer workstations, servers(including intranet or internet servers), mainframes and any integratedsystem comprising any of the above irrespective of whether theprocessing, memory, input or output of the computer is remote or local,as well as any networking interconnecting the modules of the computer.Data processing can thus be remote or distributed amongst severalprocessors at one or multiple sites. The data processing systemcomprises a data processor, such as a central processing unit (CPU),which executes instructions according to an application program. As usedherein, machines, articles of manufacture and processes are exclusive ofthe machines, manufactures, and processes employed by the United StatesPatent and Trademark Office or the European Patent Office when datarepresenting the sequence of a polypeptide or polynucleotide of thepresent invention is used for patentability searches.

The machine of the present invention includes a memory comprising datarepresenting at least one genetic sequence. As used herein, “geneticsequence” refers to the primary sequence (i.e., amino acid or nucleotidesequence) of a polynucleotide or polypeptide of the present invention.The genetic sequence can represent a partial sequence from a full-lengthprotein, genomic DNA or full-length cDNA/mRNA. Nucleic acids or proteinscomprising a genetic sequence that is identified, analyzed or modeledaccording to the present invention can be cloned or synthesized.

As those of skill in the art will be aware, the form of memory of amachine of the present invention, or the particular embodiment of thecomputer readable medium, are not critical elements of the invention andcan take a variety of forms. The memory of such a machine includes, butis not limited to, ROM or RAM or computer readable media such as, butnot limited to, magnetic media such as computer disks or hard drives ormedia such as CD-ROMs, DVDs, and the like. The memory comprising thedata representing the genetic sequence includes main memory, a registerand a cache. In some embodiments the data processing system stores thedata representing the genetic sequence in memory while processing thedata and wherein successive portions of the data are copied sequentiallyinto at least one register of the data processor for processing. Thus,the genetic sequence stored in memory can be a genetic sequence createdduring computer runtime or stored beforehand. The machine of the presentinvention includes a genetic identification, analysis or modelingprogram (discussed below) with access to the data representing thegenetic sequence. The program can be implemented in software orhardware.

The present invention further contemplates that the machine of thepresent invention will reference, directly or indirectly, a utility orfunction for the polynucleotide or polypeptide of the present invention.For example, the utility/function can be directly referenced as a dataelement in the machine and accessible by the program. Alternatively, theutility/function of the genetic can be indirectly referenced to anelectronic or written record. The function or utility of the geneticsequence can be a function or utility for the genetic sequence, or thedata representing the sequence (i.e., the genetic sequence data).

Exemplary function or utilities for the genetic sequence include: 1) itsname (per International Union of Biochemistry and Molecular Biologyrules of nomenclature) or the function of the enzyme or proteinrepresented by the genetic sequence, 2) the metabolic pathway that theprotein represented by the genetic sequence participates in, 3) thesubstrate or product or structural role of the protein represented bythe genetic sequence or 4) the phenotype (e.g., an agronomic orpharmacological trait) affected by modulating expression or activity ofthe protein represented by the genetic sequence.

The machine of the present invention also includes an output fordisplaying, printing or recording the results of the identification,analysis or modeling performed using a genetic sequence of the presentinvention. Exemplary outputs include monitors, printers or variouselectronic storage mechanisms (e.g., floppy disks, hard drives, mainmemory) which can be used to display the results or employed as a meansto input the stored data into a subsequent application or device.

In some embodiments, data representing a genetic sequence of the presentinvention is a data element within a data structure. The data structuremay be defined by the computer programs that define the processes ofidentification, modeling or analysis (see below) or it may be defined bythe programming of separate data storage and retrieval programssubroutines or systems. Thus, the present invention provides a memoryfor storing a data structure that can be accessed by a computerprogrammed to implement a process for identification, analysis ormodeling of a genetic sequence. The data structure, stored withinmemory, is associated with the data representing the genetic sequenceand reflects the underlying organization and structure of the geneticsequence to facilitate program access to data elements corresponding tological sub-components of the genetic sequence. The data structureenables the genetic sequence to be identified, analyzed or modeled. Theunderlying order and structure of a genetic sequence is datarepresenting the higher order organization of the primary sequence. Suchhigher order structures affect transcription, translation, enzymekinetics or reflects structural domains or motifs.

Exemplary logical sub-components which constitute the higher orderorganization of the genetic sequence include but are not limited to:restriction enzyme sites, endopeptidase sites, major grooves, minorgrooves, beta-sheets, alpha helices, open reading frames (ORFS), 5′untranslated regions (UTRs), 3′ UTRs, ribosome binding sites,glycosylation sites, signal peptide domains, intron-exon junctions,poly-A tails, transcription initiation sites, translation start sites,translation termination sites, methylation sites, zinc finger domains,modified amino acid sites, preproprotein-proprotein junctions,proprotein-protein junctions, transit peptide domains, single nucleotidepolymorphisms (SNPs), simple sequence repeats (SSRs), restrictionfragment length polymorphisms (RFLPs), insertion elements, transmembranespanning regions and stem-loop structures.

In another embodiment, the present invention provides a data processingsystem comprising at least one data structure in memory where the datastructure supports the accession of data representing a genetic sequenceof the present invention. The system also comprises at least one geneticidentification, analysis or modeling program which directs the executionof instructions by the system using the genetic sequence data toidentify, analyze or model at least one data element which is a logicalsub-component of the genetic sequence. An output for the processingresults is also provided.

B. Articles of Manufacture: Computer Readable Media

In one embodiment, the present invention provides a data structure in acomputer readable medium that contains data representing a geneticsequence of the present invention. The data structure is organized toreflect the logical structuring of the genetic sequence, so that thesequence can be analyzed by software programs capable of accessing thedata structure. In particular, the data structures of the presentinvention organize the genetic sequences of the present invention in amanner which allows software tools to perform an identification,analysis or modeling using logical elements of each genetic sequence.

In a further embodiment, the present invention provides amachine-readable media containing a computer program and geneticsequence data. The program provides instructions sufficient to implementa process for effecting the identification, analysis or modeling of thegenetic sequence data. The media also includes a data structurereflecting the underlying organization and structure of the data tofacilitate program access to data elements corresponding to logicalsub-components of the genetic sequence, the data structure beinginherent in the program and in the way in which the program organizesand accesses the data.

An example of a data structure resembles a layered hash table, where inone dimension the base content of the sequence is represented by astring of elements A, T, C, G and N. The direction from the 5′ end tothe 3′ end is reflected by the order from the position 0 to the positionof the length of the string minus one. Such a string, corresponding to anucleotide sequence of interest, has a certain number of substrings,each of which is delimited by the string position of its 5′ end and thestring position of its 3′ end within the parent string. In a seconddimension, each substring is associated with or pointed to one ormultiple attribute fields. Such attribute fields contain annotations tothe region on the nucleotide sequence represented by the substring.

For example, a sequence under investigation is 520 bases long andrepresented by a string named SeqTarget. There is a minor groove in the5′ upstream non-coding region from position 12 to 38, which isidentified as a binding site for an enhancer protein HM-A, which in turnwill increase the transcription of the gene represented by SeqTarget.Here, the substring is represented as (12, 38) and has the followingattributes: [upstream uncoded], [minor groove], [HM-A binding] and[increase transcription upon binding by HM-A]. Similarly, other types ofinformation can be stored and structured in this manner, such asinformation related to the whole sequence, e.g., whether the sequence isa full length viral gene, a mammalian house keeping gene or an EST fromclone X, information related to the 3′ down stream non-coding region,e.g., hair pin structure and information related to various domains ofthe coding region, e.g., Zinc finger.

This data structure is an open structure and is robust enough toaccommodate newly generated data and acquired knowledge. Such astructure is also a flexible structure. It can be trimmed down to a1-Dstring to facilitate data mining and analysis steps, such as clustering,repeat-masking and HMM analysis. Meanwhile, such a data structure alsocan extend the associated attributes into multiple dimensions. Pointerscan be established among the dimensioned attributes when needed tofacilitate data management and processing in a comprehensive genomicsknowledge base. Furthermore, such a data structure is object-oriented.Polymorphism can be represented by a family or class of sequenceobjects, each of which has an internal structure as discussed above. Thecommon traits are abstracted and assigned to the parent object, whereaseach child object represents a specific variant of the family or class.Such a data structure allows data to be efficiently retrieved, updatedand integrated by the software applications associated with the sequencedatabase and/or knowledge base.

C. Processes: Identification, Analysis, or Modeling

The present invention also provides a process of identifying, analyzing,or modeling data representing a genetic sequence of the presentinvention. The process comprises: 1) providing a machine having ahardware or software implemented genetic sequence identification,modeling, or analysis program with data representing a genetic sequence,2) executing the program while granting it access to the geneticsequence data and 3) displaying or outputting the results of theidentification, analysis, or modeling. Data structures made by theprocesses of the present invention and embodied within a computerreadable medium are also provided herein.

A further process of the present invention comprises providing a memoryembodied with data representing a genetic sequence and developing withinthe memory a data structure associated with the data and reflecting theunderlying organization and structure of the data to facilitate programaccess to data elements corresponding to logical subcomponents of thesequence. A computer is programmed with a program containinginstructions sufficient to implement the process for effecting theidentification, analysis or modeling of the genetic sequence and theprogram is executed on the computer while granting the program access tothe data and to the data structure within the memory. The programresults are outputted.

Identification, analysis, and modeling programs are well known in theart and available commercially. The program typically has at least oneapplication to: 1) identify the structural role or enzymatic function ofthe gene which the genetic sequence encodes or is translated from, 2)analyzes and identifies higher order structures within the geneticsequence or 3) model the physico-chemical properties of a geneticsequence of the present invention in a particular environment.

Included amongst the modeling/analysis tools are methods to: 1)recognize overlapping sequences (e.g., from a sequencing project) with apolynucleotide of the present invention and create an alignment called a“contig”; 2) identify restriction enzyme sites of a polynucleotide ofthe present invention; 3) identify the products of a TI ribonucleasedigestion of a polynucleotide of the present invention; 4) identify PCRprimers with minimal self-complementarity; 5) compute pairwise distancesbetween sequences in an alignment, reconstruct phylogentic trees usingdistance methods, and calculate the degree of divergence of two proteincoding regions; 6) identify patterns such as coding regions,terminators, repeats, and other consensus patterns in polynucleotides ofthe present invention; 7) identify RNA secondary structure; 8) identifysequence motifs, isoelectric point, secondary structure, hydrophobicityand antigenicity in polypeptides of the present invention; 9) translatepolynucleotides of the present invention and backtranslate polypeptidesof the present invention and 10) compare two protein or nucleic acidsequences and identifying points of similarity or dissimilarity betweenthem.

Identification of the function/utility of a genetic sequence istypically achieved by comparative analysis to a gene/protein databaseand establishing the genetic sequence as a candidate homologue (i.e.,ortholog or paralog) of a gene/protein of known function/utility.

A candidate homologue has statistically significant probability ofhaving the same biological function (e.g., catalyzes the same reaction,binds to homologous proteins/nucleic acids, has a similar structuralrole) as the reference sequence to which it is compared. Sequenceidentity/similarity is frequently employed as a criterion to identifycandidate homologues. In the same vein, genetic sequences of the presentinvention have utility in identifying homologs in animals or other plantspecies, particularly those in the family Gramineae such as, but notlimited to, sorghum, wheat or rice. Function is frequently establishedon the basis of sequence identity/similarity. Exemplary sequencecomparison systems are provided for in sequence analysis software suchas those provided by the Genetics Computer Group (Madison, Wis.) orInforMax</RTI

The present invention further provides methods for detecting apolynucleotide of the present invention in a nucleic acid samplesuspected of containing a polynucleotide of the present invention, suchas a plant cell lysate, particularly a lysate of maize. In someembodiments, a gene of the present invention or portion thereof can beamplified prior to the step of contacting the nucleic acid sample with apolynucleotide of the present invention. The nucleic acid sample iscontacted with the polynucleotide to form a hybridization complex. Thepolynucleotide hybridizes under stringent conditions to a gene encodinga polypeptide of the present invention. Formation of the hybridizationcomplex is used to detect a gene encoding a polypeptide of the presentinvention in the nucleic acid sample. Those of skill will appreciatethat an isolated nucleic acid comprising a polynucleotide of the presentinvention should lack cross-hybridizing sequences in common withnon-target genes that would yield a false positive result.

Detection of the hybridization complex can be achieved using any numberof well known methods. For example, the nucleic acid sample, or aportion thereof, may be assayed by hybridization formats including butnot limited to, solution phase, solid phase, mixed phase, or in situhybridization assays. Briefly, in solution (or liquid) phasehybridizations, both the target nucleic acid and the probe or primer arefree to interact in the reaction mixture. In solid phase hybridizationassays, probes or primers are typically linked to a solid support wherethey are available for hybridization with target nucleic in solution. Inmixed phase, nucleic acid intermediates in solution hybridize to targetnucleic acids in solution as well as to a nucleic acid linked to a solidsupport. In in situ hybridization, the target nucleic acid is liberatedfrom its cellular surroundings in such as to be available forhybridization within the cell while preserving the cellular morphologyfor subsequent interpretation and analysis. The following articlesprovide an overview of the various hybridization assay formats: Singer,et al., (1986) Biotechniques 4(3):230-250; Haase, et al., Methods inVirology, Vol. VII, pp. 189-226 (1984); Wilkinson, The theory andpractice of in situ hybridization in: In situ Hybridization, Wilkinson,Ed., IRL Press, Oxford University Press, Oxford; and Nucleic AcidHybridization: A Practical Approach, Hames and Higgins, Eds., IRL Press(1987).

Nucleic Acid Labels and Detection Methods

The means by which nucleic acids of the present invention are labeled isnot a critical aspect of the present invention and can be accomplishedby any number of methods currently known or later developed. Detectablelabels suitable for use in the present invention include any compositiondetectable by spectroscopic, radioisotopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means.

Useful labels in the present invention include biotin for staining withlabeled streptavidin conjugate, magnetic beads, fluorescent dyes (e.g.,fluorescein, Texas red, rhodamine, green fluorescent protein and thelike), radiolabels (e.g., 3H, 125I, 35S, 14C or 32P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

Nucleic acids of the present invention can be labeled by any one ofseveral methods typically used to detect the presence of hybridizednucleic acids. One common method of detection is the use ofautoradiography using probes labeled with 3H, 125I, 35S, 14C or 32P, orthe like. The choice of radioactive isotope depends on researchpreferences due to ease of synthesis, stability, and half lives of theselected isotopes. Other labels include ligands which bind to antibodieslabeled with fluorophores, chemiluminescent agents and enzymes.Alternatively, probes can be conjugated directly with labels such asfluorophores, chemiluminescent agents or enzymes. The choice of labeldepends on sensitivity required, ease of conjugation with the probe,stability requirements and available instrumentation. Labeling thenucleic acids of the present invention is readily achieved such as bythe use of labeled PCR primers.

In some embodiments, the label is simultaneously incorporated during theamplification step in the preparation of the nucleic acids. Thus, forexample, polymerase chain reaction (PCR) with labeled primers or labelednucleotides will provide a labeled amplification product. In anotherembodiment, transcription amplification using a labeled nucleotide(e.g., fluorescein-labeled UTP and/or CTP) incorporates a label into thetranscribed nucleic acids.

Non-radioactive probes are often labeled by indirect means. For example,a ligand molecule is covalently bound to the probe. The ligand thenbinds to an anti-ligand molecule which is either inherently detectableor covalently bound to a detectable signal system, such as an enzyme, afluorophore or a chemiluminescent compound. Enzymes of interest aslabels will primarily be hydrolases, such as phosphatases, esterases andglycosidases or oxidoreductases, particularly peroxidases. Fluorescentcompounds include fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, umbelliferone, etc. Chemiluminescers includeluciferin and 2,3-dihydrophthalazinediones, e.g., luminol.

Ligands and anti-ligands may be varied widely. Where a ligand has anatural anti-ligand, namely ligands such as biotin, thyroxine andcortisol, it can be used in conjunction with its labeled, naturallyoccurring anti-ligands. Alternatively, any haptenic or antigeniccompound can be used in combination with an antibody. Probes can also belabeled by direct conjugation with a label. For example, cloned DNAprobes have been coupled directly to horseradish peroxidase or alkalinephosphatase.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted light. Enzymatic labels aretypically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate and colorimetric labels are detected by simply visualizingthe colored label.

Antibodies to Proteins

Antibodies can be raised to a protein of the present invention,including individual, allelic, strain, or species variants, andfragments thereof, both in their naturally occurring (full-length) formsand in recombinant forms. Additionally, antibodies are raised to theseproteins in either their native configurations or in non-nativeconfigurations. Many methods of making antibodies are known to personsof skill. A variety of analytic methods are available to generate ahydrophilicity profile of a protein of the present invention. Suchmethods can be used to guide the artisan in the selection of peptides ofthe present invention for use in the generation or selection ofantibodies which are specifically reactive, under immunogenicconditions, to a protein of the present invention. See, e.g., Janin,(1979) Nature 277:491-492; Wolfenden, et al., (1981) Biochemistry20:849-855; Kyte and Doolite, (1982) J. Mol. Biol. 157:105-132; Rose, etal., (1985) Science 229:834838. The following discussion is presented asa general overview of the techniques available; however, one of skillwill recognize that many variations upon the following methods areknown.

A number of immunogens are used to produce antibodies specificallyreactive with a protein of the present invention. An isolatedrecombinant, synthetic or native polynucleotide of the present inventionare the preferred antigens for the production of monoclonal orpolyclonal antibodies. Polypeptides of the present invention areoptionally denatured, and optionally reduced, prior to formation ofantibodies for screening expression libraries or other assays in which aputative protein of the present invention is expressed or denatured in anon-native secondary, tertiary, or quartenary structure.

The protein of the present invention is then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies can be generated for subsequent use in immunoassays tomeasure the presence and quantity of the protein of the presentinvention.

Methods of producing polyclonal antibodies are known to those of skillin the art. In brief, an antigen, preferably a purified protein, aprotein coupled to an appropriate carrier (e.g., GST, keyhole limpethemanocyanin, etc.), or a protein incorporated into an immunizationvector such as a recombinant vaccinia virus (see, U.S. Pat. No.4,722,848) is mixed with an adjuvant and animals are immunized with themixture. The animal's immune response to the immunogen preparation ismonitored by taking test bleeds and determining the titer of reactivityto the protein of interest. When appropriately high titers of antibodyto the immunogen are obtained, blood is collected from the animal andantisera are prepared. Further fractionation of the antisera to enrichfor antibodies reactive to the protein is performed where desired (See,e.g., Coligan, Current Protocols in Immunology, Wiley/Greene, NY (1991)and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress, NY (1989)).

Antibodies, including binding fragments and single chain recombinantversions thereof, against predetermined fragments of a protein of thepresent invention are raised by immunizing animals, e.g., withconjugates of the fragments with carrier proteins as described above.Typically, the immunogen of interest is a protein of at least about 5amino acids, more typically the protein is 10 amino acids in length,preferably, 15 amino acids in length and more preferably the protein is20 amino acids in length or greater. The peptides are typically coupledto a carrier protein (e.g., as a fusion protein) or are recombinantlyexpressed in an immunization vector. Antigenic determinants on peptidesto which antibodies bind are typically 3 to 10 amino acids in length.

Monoclonal antibodies are prepared from hybrid cells secreting thedesired antibody. Monoclonals antibodies are screened for binding to aprotein from which the antigen was derived. Specific monoclonal andpolyclonal antibodies will usually have an antibody binding site with anaffinity constant for its cognate monovalent antigen at least between106-107, usually at least 108, preferably at least 109, more preferablyat least 101 and most preferably at least 101 liters/mole.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies arefound in, e.g., Basic and Clinical Immunology, 4th ed., Stites et al.,Eds., Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane, Supra; Goding, Monoclonal Antibodies:Principles and Practice, 2nd ed., Academic Press, New York, N.Y. (1986);and Kohler and Milstein, (1975) Nature 256:495-497. Summarized briefly,this method proceeds by injecting an animal with an antigen comprising aprotein of the present invention. The animal is then sacrificed andcells taken from its spleen, which are fused with myeloma cells. Theresult is a hybrid cell or “hybridoma” that is capable of reproducing invitro.

The population of hybridomas is then screened to isolate individualclones, each of which secrete a single antibody species to the antigen.In this manner, the individual antibody species obtained are theproducts of immortalized and cloned single B cells from the immuneanimal generated in response to a specific site recognized on theantigenic substance.

Other suitable techniques involve selection of libraries of recombinantantibodies in phage or similar vectors (see, e.g., Huse, et al., (1989)Science 246:1275-1281 and Ward, et al., (1989) Nature 341:544-546 andVaughan, et al., (1996) Nature Biotechnology 14:309-314). Alternatively,high avidity human monoclonal antibodies can be obtained from transgenicmice comprising fragments of the unrearranged human heavy and lightchain Ig loci (i.e., mini locus transgenic mice). Fishwild, et al.,(1996) Nature Bio Tech. 14:845-851. Also, recombinant immunoglobulinsmay be produced. See, Cabilly, U.S. Pat. No. 4,816,567 and Queen, etal., (1989) Proc. Nat'l Acad. Sci. 86:10029-10033.

The antibodies of this invention are also used for affinitychromatography in isolating proteins of the present invention. Columnsare prepared, e.g., with the antibodies linked to a solid support, e.g.,particles, such as agarose, SEPHADEX, or the like, where a cell lysateis passed through the column, washed and treated with increasingconcentrations of a mild denaturant, whereby purified protein arereleased.

The antibodies can be used to screen expression libraries for particularexpression products such as normal or abnormal protein. Usually theantibodies in such a procedure are labeled with a moiety allowing easydetection of presence of antigen by antibody binding. Antibodies raisedagainst a protein of the present invention can also be used to raiseanti-idiotypic antibodies. These are useful for detecting or diagnosingvarious pathological conditions related to the presence of therespective antigens.

Frequently, the proteins and antibodies of the present invention will belabeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles, andthe like.

Plants exhibiting an altered ethylene-dependent phenotype as comparedwith wild-type plants can be selected among other methods, by visualobservation. For example, an altered ethylene-dependent phenotype may bedetected by utilization of the “triple response.” The “triple response”consists of three distinct morphological changes in dark-grown seedlingsupon exposure to ethylene: inhibition of hypocotyl and root elongation,radial swelling of the stem and exaggeration of the apical hook. Thus, atriple response displayed in the presence of ethylene inhibitors wouldindicate one type of altered ethylene-dependent phenotype. Ethyleneaffects a vast array of agriculturally important plant processes,including fruit ripening, flower and leaf senescence and leafabscission. The ability to control the sensitivity of plants to ethylenecould thus significantly improve the quality and longevity of manycrops. The invention includes plants produced by the method of theinvention, as well as plant tissue and seeds.

“Stacking” of Constructs and Traits

In certain embodiments, the nucleic acid sequences of the presentinvention can be used in combination (“stacked”) with otherpolynucleotide sequences of interest in order to create plants with adesired phenotype. The polynucleotides of the present invention may bestacked with any gene or combination of genes, and the combinationsgenerated can include multiple copies of any one or more of thepolynucleotides of interest. The desired combination may affect one ormore traits; that is, certain combinations may be created for modulationof gene expression affecting ACC synthase activity and/or ethyleneproduction. Other combinations may be designed to produce plants with avariety of desired traits, including but not limited to traits desirablefor animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529);balanced amino acids (e.g., hordothionins (U.S. Pat. Nos. 5,990,389;5,885,801; 5,885,802 and 5,703,409); barley high lysine (Williamson, etal., (1987) Eur. J. Biochem. 165:99-106 and WO 1998/20122) and highmethionine proteins (Pedersen, et al., (1986) J. Biol. Chem. 261:6279;Kirihara, et al., (1988) Gene 71:359 and Musumura, et al., (1989) PlantMol. Biol. 12:123)); increased digestibility (e.g., modified storageproteins (U.S. patent application Ser. No. 10/053,410, filed Nov. 7,2001) and thioredoxins (U.S. patent application Ser. No. 10/005,429,filed Dec. 3, 2001)), the disclosures of which are herein incorporatedby reference. The polynucleotides of the present invention can also bestacked with traits desirable for insect, disease or herbicideresistance (e.g., Bacillus thuringiensis toxic proteins (U.S. Pat. Nos.5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser, et al.,(1986) Gene 48:109); lectins (Van Damme, et al., (1994) Plant Mol. Biol.24:825); fumonisin detoxification genes (U.S. Pat. No. 5,792,931);avirulence and disease resistance genes (Jones, et al., (1994) Science266:789; Martin, et al., (1993) Science 262:1432; Mindrinos, et al.,(1994) Cell 78:1089); acetolactate synthase (ALS) mutants that lead toherbicide resistance such as the S4 and/or Hra mutations; inhibitors ofglutamine synthase such as phosphinothricin or basta (e.g., bar gene)and glyphosate resistance (EPSPS gene)) and traits desirable forprocessing or process products such as high oil (e.g., U.S. Pat. No.6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat.No. 5,952,544; WO 1994/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE) and starch debranching enzymes (SDBE)); and polymers orbioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase,polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert,et al., (1988) J. Bacteriol. 170:5837-5847) facilitate expression ofpolyhydroxyalkanoates (PHAs)), the disclosures of which are hereinincorporated by reference. One could also combine the polynucleotides ofthe present invention with polynucleotides affecting agronomic traitssuch as male sterility (e.g., see, U.S. Pat. No. 5,583,210), stalkstrength, flowering time, or transformation technology traits such ascell cycle regulation or gene targeting (e.g., WO 1999/61619; WO2000/17364; WO 1999/25821), the disclosures of which are hereinincorporated by reference.

These stacked combinations can be created by any method, including butnot limited to cross breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the traits are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences of interest can be driven bythe same promoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of a polynucleotide of interest. This may be accompanied byany combination of other suppression cassettes or over-expressioncassettes to generate the desired combination of traits in the plant.

Use in Breeding Methods

The transformed plants of the invention may be used in a plant breedingprogram. The goal of plant breeding is to combine, in a single varietyor hybrid, various desirable traits. For field crops, these traits mayinclude, for example, resistance to diseases and insects, tolerance toheat and drought, reduced time to crop maturity, greater yield andbetter agronomic quality. With mechanical harvesting of many crops,uniformity of plant characteristics such as germination and standestablishment, growth rate, maturity and plant and ear height isdesirable. Traditional plant breeding is an important tool in developingnew and improved commercial crops. This invention encompasses methodsfor producing a maize plant by crossing a first parent maize plant witha second parent maize plant wherein one or both of the parent maizeplants is a transformed plant displaying a staygreen phenotype, asterility phenotype, a crowding resistance phenotype, or the like, asdescribed herein.

Plant breeding techniques known in the art and used in a maize plantbreeding program include, but are not limited to, recurrent selection,bulk selection, mass selection, backcrossing, pedigree breeding, openpollination breeding, restriction fragment length polymorphism enhancedselection, genetic marker enhanced selection, doubled haploids andtransformation. Often combinations of these techniques are used.

The development of maize hybrids in a maize plant breeding programrequires, in general, the development of homozygous inbred lines, thecrossing of these lines and the evaluation of the crosses. There aremany analytical methods available to evaluate the result of a cross. Theoldest and most traditional method of analysis is the observation ofphenotypic traits. Alternatively, the genotype of a plant can beexamined.

A genetic trait which has been engineered into a particular maize plantusing transformation techniques can be moved into another line usingtraditional breeding techniques that are well known in the plantbreeding arts. For example, a backcrossing approach is commonly used tomove a transgene from a transformed maize plant to an elite inbred line,and the resulting progeny would then comprise the transgene(s). Also, ifan inbred line was used for the transformation, then the transgenicplants could be crossed to a different inbred in order to produce atransgenic hybrid maize plant. As used herein, “crossing” can refer to asimple X by Y cross, or the process of backcrossing, depending on thecontext.

The development of a maize hybrid in a maize plant breeding programinvolves three steps: (1) the selection of plants from various germplasmpools for initial breeding crosses; (2) the selfing of the selectedplants from the breeding crosses for several generations to produce aseries of inbred lines, which, while different from each other, breedtrue and are highly uniform and (3) crossing the selected inbred lineswith different inbred lines to produce the hybrids. During theinbreeding process in maize, the vigor of the lines decreases. Vigor isrestored when two different inbred lines are crossed to produce thehybrid. An important consequence of the homozygosity and homogeneity ofthe inbred lines is that the hybrid created by crossing a defined pairof inbreds will always be the same. Once the inbreds that give asuperior hybrid have been identified, the hybrid seed can be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained.

Transgenic plants of the present invention may be used to produce, e.g.,a single cross hybrid, a three-way hybrid or a double cross hybrid. Asingle cross hybrid is produced when two inbred lines are crossed toproduce the F1 progeny. A double cross hybrid is produced from fourinbred lines crossed in pairs (A×B and C×D) and then the two F1 hybridsare crossed again (A×B)×(C×D). A three-way cross hybrid is produced fromthree inbred lines where two of the inbred lines are crossed (A×B) andthen the resulting F1 hybrid is crossed with the third inbred (A×B)×C.Much of the hybrid vigor and uniformity exhibited by F1 hybrids is lostin the next generation (F2). Consequently, seed produced by hybrids isconsumed rather than planted.

Antibodies

The polypeptides of the invention can be used to produce antibodiesspecific for the polypeptides of SEQ ID NO: 2, 4, 6, 8 or 10 andconservative variants thereof. Antibodies specific for, e.g., SEQ ID NO:2, 4, 6, 8 or 10 and related variant polypeptides are useful, e.g., forscreening and identification purposes, e.g., related to the activity,distribution and expression of ACC synthase.

Antibodies specific for the polypeptides of the invention can begenerated by methods well known in the art. Such antibodies can include,but are not limited to, polyclonal, monoclonal, chimeric, humanized,single chain, Fab fragments and fragments produced by a Fab expressionlibrary.

Polypeptides do not require biological activity for antibody production.The full length polypeptide, subsequences, fragments or oligopeptidescan be antigenic. Peptides used to induce specific antibodies typicallyhave an amino acid sequence of at least about 10 amino acids and oftenat least 15 or 20 amino acids. Short stretches of a polypeptide, e.g.,selected from among SEQ ID NO: 2, 4, 6, 8 or 10, can be fused withanother protein, such as keyhole limpet hemocyanin and antibody producedagainst the chimeric molecule.

Numerous methods for producing polyclonal and monoclonal antibodies areknown to those of skill in the art and can be adapted to produceantibodies specific for the polypeptides of the invention, e.g.,corresponding to SEQ ID NO: 2, 4, 6, 8 or 10. See, e.g., Coligan (1991)Current Protocols in Immunology Wiley/Greene, NY; and Harlow and Lane(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY;Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) LangeMedical Publications, Los Altos, Calif., and references cited therein;Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.)Academic Press, New York, N.Y.; Fundamental Immunology, e.g., 4thEdition (or later), W. E. Paul (ed.), Raven Press, N.Y. (1998) andKohler and Milstein, (1975) Nature 256:495-497. Other suitabletechniques for antibody preparation include selection of libraries ofrecombinant antibodies in phage or similar vectors. See, Huse, et al.,(1989) Science 246:1275-1281 and Ward, et al., (1989) Nature341:544-546. Specific monoclonal and polyclonal antibodies and antiserawill usually bind with a K_(D) of at least about 0.1 μM, preferably atleast about 0.01 μM or better and most typically and preferably, 0.001μM or better.

Kits for Modulating Plant Stress Response

Certain embodiments of the invention can optionally be provided to auser as a kit. For example, a kit of the invention can contain one ormore nucleic acid, polypeptide, antibody, diagnostic nucleic acid orpolypeptide, e.g., antibody, probe set, e.g., as a cDNA microarray, oneor more vector and/or cell line described herein. Most often, the kit ispackaged in a suitable container. The kit typically further comprisesone or more additional reagents, e.g., substrates, labels, primers, orthe like for labeling expression products, tubes and/or otheraccessories, reagents for collecting samples, buffers, hybridizationchambers, cover slips, etc. The kit optionally further comprises aninstruction set or user manual detailing preferred methods of using thekit components for discovery or application of gene sets. When usedaccording to the instructions, the kit can be used, e.g., for evaluatingexpression or polymorphisms in a plant sample, e.g., for evaluating ACCsynthase, ethylene production, stress response potential, crowdingresistance potential, sterility, etc. Alternatively, the kit can be usedaccording to instructions for using at least one ACC synthasepolynucleotide sequence to control ethylene production in a plant.

Other Nucleic Acid and Protein Assays

In the context of the invention, nucleic acids and/or proteins aremanipulated according to well known molecular biology methods. Detailedprotocols for numerous such procedures are described in, e.g., inAusubel, et al., Current Protocols in Molecular Biology (supplementedthrough 2004) John Wiley & Sons, New York (“Ausubel”); Sambrook, et al.,Molecular Cloning-A Laboratory Manual (2nd Ed.), Vol. 1 3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”) andBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif.(“Berger”).

In addition to the above references, protocols for in vitroamplification techniques, such as the polymerase chain reaction (PCR),the ligase chain reaction (LCR), Qβ-replicase amplification and otherRNA polymerase mediated techniques (e.g., NASBA), useful, e.g., foramplifying polynucleotides of the invention, are found in Mullis, etal., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methodsand Applications (Innis et al. eds) Academic Press Inc. San Diego,Calif. (1990) (“Innis”); Arnheim and Levinson, (1990) C&EN 36; TheJournal Of NIH Research (1991) 3:81; Kwoh, et al., (1989) Proc Natl AcadSci USA 86:1173; Guatelli, et al., (1990) Proc Natl Acad Sci USA87:1874; Lomell, et al., (1989) J. Clin. Chem. 35:1826; Landegren, etal., (1988) Science 241:1077; Van Brunt, (1990) Biotechnology 8:291; Wuand Wallace, (1989) Gene 4:560; Barringer, et al., (1990) Gene 89:117and Sooknanan and Malek, (1995) Biotechnology 13:563. Additionalmethods, useful for cloning nucleic acids in the context of theinvention, include Wallace, et al., U.S. Pat. No. 5,426,039. Improvedmethods of amplifying large nucleic acids by PCR are summarized inCheng, et al., (1994) Nature 369:684 and the references therein.

Certain polynucleotides of the invention can be synthesized utilizingvarious solid-phase strategies involving mononucleotide- and/ortrinucleotide-based phosphoramidite coupling chemistry. For example,nucleic acid sequences can be synthesized by the sequential addition ofactivated monomers and/or trimers to an elongating polynucleotide chain.See, e.g., Caruthers, et al., (1992) Meth Enzymol 211:3. In lieu ofsynthesizing the desired sequences, essentially any nucleic acid can becustom ordered from any of a variety of commercial sources, such as TheMidland Certified Reagent Company (mcrc@oligos.com) (Midland, Tex.), TheGreat American Gene Company (available on the World Wide Web atgenco.com) (Ramona, Calif.), ExpressGen, Inc. (available on the WorldWide Web at expressgen.com) (Chicago III.), Operon Technologies, Inc.(available on the World Wide Web at operon.com) (Alameda Calif.), andmany others.

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

TABLE 1 Sequences in Sequence Listing SEQ ID NO PP/NT DESCRIPTION 1nucleotide ZM-ETO1-1 cfp3n.pk009.o19.f.FIS 2 polypeptide ZM-ETO1-1cfp3n.pk009.o19.f.FIS 3 nucleotide ZM-ETO1-2 cfp6n.pk073.o21.FIS 4polypeptide ZM-ETO1-2cfp6n.pk073.o21.FIS 5 nucleotide ZM-ETO1-3cta1.pk0036.f.FIS 6 polypeptide ZM-ETO1-3 cta1.pk0036.f +cfp1n.pk047.e9a.FIS 7 nucleotide ZM-ETO1-4 cfp7n.pk074.p17.FIS 8polypeptide ZM-ETO1-4 cfp7n.pk074.p17.FIS 9 nucleotide GM-ETO1-1PSO415110 genomic 10 polypeptide GM-ETO1-1 PSO415110 11 polypeptideN-Terminal Domain of ETO1 (Consensus) 12 polypeptide C-Terminal Domainof ETO1 (Consensus) 13 nucleotide ASal-A20 oligonucleotide

EXAMPLES Example 1 Construction of cDNA Libraries Total RNA Isolation

Total RNA for SEQ ID NO: 1, 3, 5 and 7 was obtained from maize genotypeHill (Armstrong and Phillips, (1988) Crop Sci. 28:363-369) and fromnight harvested leaf tissue at the V8-V10 stage of maize genotype B75.Total RNA for SEQ ID NO: 9 was obtained from soybean. The total RNA wasisolated from the maize and soybean tissues with TRIzol Reagent (LifeTechnology Inc. Gaithersburg, Md.) using a modification of the guanidineisothiocyanate/acid-phenol procedure described by Chomczynski and Sacchi(Chomczynski and Sacchi, (1987) Anal. Biochem. 162:156). In brief, planttissue samples were pulverized in liquid nitrogen before the addition ofthe TRIzol Reagent and then were further homogenized with a mortar andpestle. Addition of chloroform followed by centrifugation was conductedfor separation of an aqueous phase and an organic phase. The total RNAwas recovered by precipitation with isopropyl alcohol from the aqueousphase.

Poly (A)+ RNA Isolation

The selection of poly (A)+ RNA from total RNA was performed usingPolyATact system (Promega Corporation, Madison, Wis.). In brief,biotinylated oligo(dT) primers were used to hybridize to the 3′ poly (A)tails on mRNA. The hybrids were captured using streptavidin coupled toparamagnetic particles and a magnetic separation stand. The mRNA waswashed at high stringency conditions and eluted by RNase-free deionizedwater. cDNA Library Construction cDNA synthesis was performed andunidirectional cDNA libraries were constructed using the SuperScriptPlasmid System (Life Technology Inc. Gaithersburg, Md.). The firststrand of cDNA was synthesized by priming an oligo(dT) primer containinga Not I site.

The reaction was catalyzed by SuperScript Reverse Transcriptase II at45° C. The second strand of cDNA was labeled with alpha-32P-dCTP and aportion of the reaction was analyzed by agarose gel electrophoresis todetermine cDNA sizes. cDNA molecules smaller than 500 base pairs andunligated adapters were removed by Sephacryl-5400 chromatography. Theselected cDNA molecules were ligated into pSPORTI vector in between ofNot I and Sal I sites.

TABLE 2 cDNAs, Corresponding Sequence Identifiers, and Sourcecfp3n.pk009.o19.f SEQ ID 1 & 2 Maize Ear, pooled V10-V14-v16- VT,Full-length enriched normalized cfp6n.pk073.o21.f SEQ ID 3 & 4 MaizeLeaf and Seed pooled, Full- length enriched normalized cfp6n.pk003.j6SEQ ID 5 & 6 Maize Leaf and Seed pooled, Full- length enrichednormalized cfp7n.pk074.p17 SEQ ID 7 & 8 Maize Root, Pooled stages, Full-length enriched, normalized sfl1.pk0066.b1 SEQ ID 9 & 10 Soybean(Glycine max L.) immature flowerBased on the sequence comparison of the soybean and maize sequences twodomains that are highly conserved across all of the sequences wereidentified: one N-terminal and one C-terminal.

N-TERMINAL DOMAIN (SEQ ID NO: 11)F-X8-C-X-R-X3-A-X-L-S-X-P-X4-L-X-G-X-F-X-E-X17-M-X6-S-X16-L-X2-A-X2-F-C-C-X2-L-K-X2-C-X3-L-X8-A-X8- E-X5-L-X3-CLQC-TERMINAL DOMAIN (SEQ ID NO: 12)W-S-X-V-D-D-X2-S-L-X-V-X3-M-L-X8-L-X-F-R-Q-S-L-L-L-L-R-L-N-C-X3-A-M-R-X-L-X2-A-X8-E-R-L-V-Y-E-G-W-X-L-Y-D-X-G-X3-E-X-L-X-K-A-X3-I-X3-R-S-F-E-A-X-F-L-X-A-Y-X-L-X5-D-X6-V-X3-L-X2-A-X2-C-X2-D-X-L-R-K-G-Q-A-X-N-N-X-G-X2-Y-X5-L-D-X-A-X3-Y-X2-A-X4-H-X-R-A-X-Q-G-L-A-R-V-X2-L-X-N-X4-A-X2-E-M-T-X-L-X-E-X5-A-X-A-Y-E-K-R-S-E-Y-X2-R-X5-D-L-X5-L-D-P-X-R-X-Y-P-Y-R-Y-R-A-A-V-L-M-D

Example 2 cDNA Sequencing and Library Subtraction Sequencing TemplatePreparation

Individual colonies were picked and DNA was prepared either by PCR withM13 forward primers and M13 reverse primers, or by plasmid isolation.All the cDNA clones were sequenced using M13 reverse primers.

Q-bot Subtraction Procedure cDNA libraries subjected to the subtractionprocedure were plated out on 22×22 cm² agar plate at density of about3,000 colonies per plate. The plates were incubated in a 37° C.incubator for 12-24 hours. Colonies were picked into 384-well plates bya robot colony picker, Q-bot (GENETIX Limited). These plates wereincubated overnight at 37° C.

Once sufficient colonies were picked, they were pinned onto 22×22 cm²nylon membranes using Q-bot. Each membrane contained 9,216 colonies or36,864 colonies. These membranes were placed onto agar plate withappropriate antibiotic. The plates were incubated at 37° C. forovernight.

After colonies were recovered on the second day, these filters wereplaced on filter paper pre-wetted with denaturing solution for fourminutes, then were incubated on top of a boiling water bath foradditional four minutes. The filters were then placed on filter paperpre-wetted with neutralizing solution for four minutes. After excesssolution was removed by placing the filters on dry filter papers for oneminute, the colony side of the filters were place into Proteinase Ksolution, incubated at 37° C. for 40-50 minutes. The filters were placedon dry filter papers to dry overnight. DNA was then cross-linked tonylon membrane by UV light treatment.

Colony hybridization was conducted as described by Sambrook, et al., (inMolecular Cloning: A laboratory Manual, 2nd Edition). The followingprobes were used in colony hybridization:

-   -   1. First strand cDNA from the same tissue as the library was        made from to remove the most redundant clones.    -   2. 48-192 most redundant cDNA clones from the same library based        on previous sequencing data.    -   3. 192 most redundant cDNA clones in the entire maize sequence        database.    -   4. ASal-A20 oligonucleotide: TCG ACC CAC GCG TCC GAA AAA AAA AAA        AAA AAA AAA (SEQ ID NO: 13), removes clones containing a poly A        tail but no cDNA.    -   5. cDNA clones derived from rRNA.

The image of the autoradiography was scanned into computer and thesignal intensity and cold colony addresses of each colony was analyzed.Re-arraying of cold colonies from 384 well plates to 96 well plates wasconducted using Q-bot.

Example 3 Homology Search

This example describes identification of the gene from a computerhomology search. Gene identities were determined by conducting BLAST(Basic Local Alignment Search Tool; Altschul, et al., (1993) J. Mol.Biol. 215:403-410) searches under default parameters for similarity tosequences contained in the BLAST “nr” database (comprising allnon-redundant GenBank CDS translations, sequences derived from the3-dimensional structure Brookhaven Protein Data Bank, the last majorrelease of the SWISS-PROT protein sequence database, EMBL and DDBJdatabases). The cDNA sequences were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm.

The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States, (1993) NatureGenetics 3:266-272) provided by the NCBI. In some cases, the sequencingdata from two or more clones containing overlapping segments of DNA wereused to construct contiguous DNA sequences.

Example 4 Transformation and Regeneration of Transgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing the ETO1 sequence operably linked to the a promotersuch as a drought-inducible promoter RAB17 promoter (Vilardell, et al.,(1990) Plant Mol Biol 14:423-432), a constitutive promoter, a femalepreferred promoter, such as ZM-ADF4 (US Patent Application PublicationNumber 2009/0094713) or EEP1 (US Patent Application Publication Number2004/0237147) or a root specific promoter and the selectable marker geneMO-PAT, which confers resistance to the herbicide Bialaphos or the BARselectable marker. Alternatively, the selectable marker gene is providedon a separate plasmid. Transformation is performed as follows. Mediarecipes follow below.

Preparation of Target Tissue

The ears are husked and surface sterilized in 30% Clorox® bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5-cm target zone in preparation forbombardment.

Preparation of DNA

A plasmid vector comprising the ETO1 sequence operably linked to anubiquitin promoter is made. This plasmid DNA plus plasmid DNA containinga PAT selectable marker is precipitated onto 1.1 μm (average diameter)tungsten pellets using a CaCl₂ precipitation procedure as follows:

100 μl prepared tungsten particles in water

10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)

100 μl 2.5 M CaCl₂

10 μl 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol and centrifugedfor 30 seconds. Again the liquid is removed and 105 μl 100% ethanol isadded to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment

The sample plates are bombarded at level #4 in a particle gun. Allsamples receive a single shot at 650 PSI, with a total of ten aliquotstaken from each tube of prepared particles/DNA.

Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for increased drought tolerance. Assaysto measure improved drought tolerance are routine in the art andinclude, for example, increased kernel set under drought conditions whencompared to control maize plants under identical environmentalconditions. Alternatively, the transformed plants can be monitored for amodulation in meristem development (e.g., a decrease in spikeletformation on the ear). See, for example, Bruce, et al., (2002) Journalof Experimental Botany 53:13-25.

Bombardment and Culture Media

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D and 2.88 g/l L-proline(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H₂O) and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/l N6 basalsalts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511),0.5 mg/l thiamine HCl, 30.0 g/l sucrose and 2.0 mg/l 2,4-D (brought tovolume with D-I H₂O following adjustment to pH 5.8 with KOH); 3.0 g/lGelrite (added after bringing to volume with D-I H₂O) and 0.85 mg/lsilver nitrate and 3.0 mg/l bialaphos (both added after sterilizing themedium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL and 0.40 g/l glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog, (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite (addedafter bringing to volume with D-I H₂O) and 1.0 mg/l indoleacetic acidand 3.0 mg/l bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinicacid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL and 0.40 g/lglycine brought to volume with polished D-I H₂O), 0.1 g/l myo-inositoland 40.0 g/l sucrose (brought to volume with polished D-I H₂O afteradjusting pH to 5.6) and 6 g/l bacto-agar (added after bringing tovolume with polished D-I H₂O), sterilized and cooled to 60° C.

Example 5 Agrobacterium-Mediated Transformation

For Agrobacterium-mediated transformation of maize with an expressionconstruct with the ETO1 sequence of the present invention, preferablythe method of Zhao is employed (U.S. Pat. No. 5,981,840 and PCT PatentApplication Publication WO 1998/32326; the contents of which are herebyincorporated by reference). Briefly, immature embryos are isolated frommaize and the embryos contacted with a suspension of Agrobacterium,where the bacteria are capable of transferring the ETO1 sequences to atleast one cell of at least one of the immature embryos (step 1: theinfection step). In this step the immature embryos are preferablyimmersed in an Agrobacterium suspension for the initiation ofinoculation. The embryos are co-cultured for a time with theAgrobacterium (step 2: the co-cultivation step). Preferably the immatureembryos are cultured on solid medium following the infection step.Following this co-cultivation period an optional “resting” step iscontemplated. In this resting step, the embryos are incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). Preferably the immature embryosare cultured on solid medium with antibiotic, but without a selectingagent, for elimination of Agrobacterium and for a resting phase for theinfected cells. Next, inoculated embryos are cultured on mediumcontaining a selective agent and growing transformed callus is recovered(step 4: the selection step). Preferably, the immature embryos arecultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step) and preferably calli grownon selective medium are cultured on solid medium to regenerate theplants. Plants are monitored and scored for a modulation in meristemdevelopment: for instance, alterations of size and appearance of theshoot and floral meristems and/or increased yields of leaves, flowersand/or fruits.

Example 6 Soybean Embryo Transformation

Soybean embryos are bombarded with a plasmid containing an ETO1 sequenceoperably linked to an ubiquitin or other constitutive promoter asfollows. To induce somatic embryos, cotyledons, 3-5 mm in lengthdissected from surface-sterilized, immature seeds of the soybeancultivar A2872, are cultured in the light or dark at 26° C. on anappropriate agar medium for six to ten weeks. Somatic embryos producingsecondary embryos are then excised and placed into a suitable liquidmedium. After repeated selection for clusters of somatic embryos thatmultiplied as early, globular-staged embryos, the suspensions aremaintained as described below.

Soybean embryogenic suspension cultures can be maintained in 35 mlliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 ml ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein, et al., (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz, et al., (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising an ETO1 encodingsequence operably linked to the ubiquitin promoter can be isolated as arestriction fragment. This fragment can then be inserted into a uniquerestriction site of the vector carrying the marker gene.

To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media and eleven to twelve days post-bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 7 Sunflower Meristem Tissue Transformation

Sunflower meristem tissues are transformed with an expression cassettecontaining an ETO1 sequence operably linked to a ubiquitin promoter asfollows (see also, European Patent Number EP 0 486233, hereinincorporated by reference and Malone-Schoneberg, et al., (1994) PlantScience 103:199-207). Mature sunflower seed (Helianthus annuus L.) aredehulled using a single wheat-head thresher. Seeds are surfacesterilized for 30 minutes in a 20% Clorox® bleach solution with theaddition of two drops of Tween® 20 per 50 ml of solution. The seeds arerinsed twice with sterile distilled water.

Split embryonic axis explants are prepared by a modification ofprocedures described by Schrammeijer, et al. (Schrammeijer, et al.,(1990) Plant Cell Rep. 9:55-60). Seeds are imbibed in distilled waterfor 60 minutes following the surface sterilization procedure. Thecotyledons of each seed are then broken off, producing a clean fractureat the plane of the embryonic axis. Following excision of the root tip,the explants are bisected longitudinally between the primordial leaves.The two halves are placed, cut surface up, on GBA medium consisting ofMurashige and Skoog mineral elements (Murashige, et al., (1962) Physiol.Plant., 15:473-497), Shepard's vitamin additions (Shepard, (1980) inEmergent Techniques for the Genetic Improvement of Crops (University ofMinnesota Press, St. Paul, Minn.) 0, 40 mg/l adenine sulfate, 30 WIsucrose, 0.5 mg/l 6-benzyl-aminopurine (BAP), 0.25 mg/l indole-3-aceticacid (IAA), 0.1 mg/l gibberellic acid (GA3), pH 5.6 and 8 WI Phytagar.

The explants are subjected to microprojectile bombardment prior toAgrobacterium treatment (Bidney, et al., (1992) Plant Mol. Biol.18:301-313). Thirty to forty explants are placed in a circle at thecenter of a 60×20 mm plate for this treatment. Approximately 4.7 mg of1.8 mm tungsten microprojectiles are resuspended in 25 ml of sterile TEbuffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are usedper bombardment. Each plate is bombarded twice through a 150 mm nytexscreen placed 2 cm above the samples in a PDS 1000® particleacceleration device.

Disarmed Agrobacterium tumefaciens strain EHA105 is used in alltransformation experiments. A binary plasmid vector comprising theexpression cassette that contains the ETO1 gene operably linked to theubiquitin promoter is introduced into Agrobacterium strain EHA105 viafreeze-thawing as described by Holsters, et al., (1978) Mol. Gen. Genet.163:181-187. This plasmid further comprises a kanamycin selectablemarker gene (i.e., nptII). Bacteria for plant transformation experimentsare grown overnight (28° C. and 100 RPM continuous agitation) in liquidYEP medium (10 gm/l yeast extract, 10 gm/l Bactopeptone, and 5 gm/lNaCl, pH 7.0) with the appropriate antibiotics required for bacterialstrain and binary plasmid maintenance. The suspension is used when itreaches an OD₆₀₀ of about 0.4 to 0.8. The Agrobacterium cells arepelleted and resuspended at a final OD₆₀₀ of 0.5 in an inoculationmedium comprised of 12.5 mM MES pH 5.7, 1 gm/l NH₄Cl, and 0.3 gm/lMgSO₄.

Freshly bombarded explants are placed in an Agrobacterium suspension,mixed, and left undisturbed for 30 minutes. The explants are thentransferred to GBA medium and co-cultivated, cut surface down, at 26° C.and 18-hour days. After three days of co-cultivation, the explants aretransferred to 374B (GBA medium lacking growth regulators and a reducedsucrose level of 1%) supplemented with 250 mg/l cefotaxime and 50 mg/lkanamycin sulfate. The explants are cultured for two to five weeks onselection and then transferred to fresh 374B medium lacking kanamycinfor one to two weeks of continued development. Explants withdifferentiating, antibiotic-resistant areas of growth that have notproduced shoots suitable for excision are transferred to GBA mediumcontaining 250 mg/l cefotaxime for a second 3-day phytohormonetreatment. Leaf samples from green, kanamycin-resistant shoots areassayed for the presence of NPTII by ELISA and for the presence oftransgene expression by assaying for a modulation in meristemdevelopment (i.e., an alteration of size and appearance of shoot andfloral meristems).

NPTII-positive shoots are grafted to Pioneer® hybrid 6440 in vitro-grownsunflower seedling rootstock. Surface sterilized seeds are germinated in48-0 medium (half-strength Murashige and Skoog salts, 0.5% sucrose, 0.3%gelrite, pH 5.6) and grown under conditions described for explantculture. The upper portion of the seedling is removed, a 1 cm verticalslice is made in the hypocotyl and the transformed shoot inserted intothe cut. The entire area is wrapped with parafilm to secure the shoot.Grafted plants can be transferred to soil following one week of in vitroculture. Grafts in soil are maintained under high humidity conditionsfollowed by a slow acclimatization to the greenhouse environment.Transformed sectors of T₀ plants (parental generation) maturing in thegreenhouse are identified by NPTII ELISA and/or by ETO1 activityanalysis of leaf extracts while transgenic seeds harvested fromNPTII-positive T₀ plants are identified by ETO1 analysis of smallportions of dry seed cotyledon.

An alternative sunflower transformation protocol allows the recovery oftransgenic progeny without the use of chemical selection pressure. Seedsare dehulled and surface-sterilized for 20 minutes in a 20% Cloroxbleach solution with the addition of two to three drops of Tween 20 per100 ml of solution, then rinsed three times with distilled water.Sterilized seeds are imbibed in the dark at 26° C. for 20 hours onfilter paper moistened with water. The cotyledons and root radical areremoved, and the meristem explants are cultured on 374E (GBA mediumconsisting of MS salts, Shepard vitamins, 40 mg/l adenine sulfate, 3%sucrose, 0.5 mg/l 6-BAP, 0.25 mg/l IAA, 0.1 mg/l GA and 0.8% Phytagar atpH 5.6) for 24 hours under the dark. The primary leaves are removed toexpose the apical meristem, around 40 explants are placed with theapical dome facing upward in a 2 cm circle in the center of 374M (GBAmedium with 1.2% Phytagar) and then cultured on the medium for 24 hoursin the dark.

Approximately 18.8 mg of 1.8 μm tungsten particles are resuspended in150 μl absolute ethanol. After sonication, 8 μl of it is dropped on thecenter of the surface of macrocarrier. Each plate is bombarded twicewith 650 psi rupture discs in the first shelf at 26 mm of Hg helium gunvacuum.

The plasmid of interest is introduced into Agrobacterium tumefaciensstrain EHA105 via freeze thawing as described previously. The pellet ofovernight-grown bacteria at 28° C. in a liquid YEP medium (10 g/l yeastextract, 10 g/l Bactopeptone, and 5 g/l NaCl, pH 7.0) in the presence of50 μg/l kanamycin is resuspended in an inoculation medium (12.5 mM 2-mM2-(N-morpholino) ethanesulfonic acid, MES, 1 g/l NH₄Cl and 0.3 g/l MgSO₄at pH 5.7) to reach a final concentration of 4.0 at OD₆₀₀.Particle-bombarded explants are transferred to GBA medium (374E), and adroplet of bacteria suspension is placed directly onto the top of themeristem. The explants are co-cultivated on the medium for 4 days, afterwhich the explants are transferred to 374C medium (GBA with 1% sucroseand no BAP, IAA, GA3 and supplemented with 250 μg/ml cefotaxime). Theplantlets are cultured on the medium for about two weeks under 16-hourday and 26° C. incubation conditions.

Explants (around 2 cm long) from two weeks of culture in 374C medium arescreened for a modulation in meristem development (i.e., an alterationof size and appearance of shoot and floral meristems). After positiveexplants are identified, those shoots that fail to exhibit modified ETO1activity are discarded, and every positive explant is subdivided intonodal explants. One nodal explant contains at least one potential node.The nodal segments are cultured on GBA medium for three to four days topromote the formation of auxiliary buds from each node. Then they aretransferred to 374C medium and allowed to develop for an additional fourweeks. Developing buds are separated and cultured for an additional fourweeks on 374C medium. Pooled leaf samples from each newly recoveredshoot are screened again by the appropriate protein activity assay. Atthis time, the positive shoots recovered from a single node willgenerally have been enriched in the transgenic sector detected in theinitial assay prior to nodal culture.

Recovered shoots positive for modified ETO1 expression are grafted toPioneer hybrid 6440 in vitro-grown sunflower seedling rootstock. Therootstocks are prepared in the following manner. Seeds are dehulled andsurface-sterilized for 20 minutes in a 20% Clorox® bleach solution withthe addition of two to three drops of Tween 20 per 100 ml of solution,and are rinsed three times with distilled water. The sterilized seedsare germinated on the filter moistened with water for three days, thenthey are transferred into 48 medium (half-strength MS salt, 0.5%sucrose, 0.3% gelrite pH 5.0) and grown at 26° C. under the dark forthree days, then incubated at 16-hour-day culture conditions. The upperportion of selected seedling is removed, a vertical slice is made ineach hypocotyl, and a transformed shoot is inserted into a V-cut. Thecut area is wrapped with parafilm. After one week of culture on themedium, grafted plants are transferred to soil. In the first two weeks,they are maintained under high humidity conditions to acclimatize to agreenhouse environment.

Example 8 Rice Tissue Transformation

One method for transforming DNA into cells of higher plants that isavailable to those skilled in the art is high-velocity ballisticbombardment using metal particles coated with the nucleic acidconstructs of interest (see, Klein, et al., (1987) Nature (London)327:70-73 and see, U.S. Pat. No. 4,945,050). A Biolistic PDS-1000/He(BioRAD Laboratories, Hercules, Calif.) is used for thesecomplementation experiments. The particle bombardment technique is usedto transform the ETO1 mutants and wild type rice with DNA fragments

The bacterial hygromycin B phosphotransferase (Hpt II) gene fromStreptomyces hygroscopicus that confers resistance to the antibiotic isused as the selectable marker for rice transformation. In the vector,pML18, the Hpt II gene was engineered with the 35S promoter fromCauliflower Mosaic Virus and the termination and polyadenylation signalsfrom the octopine synthase gene of Agrobacterium tumefaciens. pML18 wasdescribed in WO 1997/47731, which was published on Dec. 18, 1997, thedisclosure of which is hereby incorporated by reference.

Embryogenic callus cultures derived from the scutellum of germinatingrice seeds serve as source material for transformation experiments. Thismaterial is generated by germinating sterile rice seeds on a callusinitiation media (MS salts, Nitsch and Nitsch vitamins, 1.0 mg/l 2,4-Dand 10 μM AgNO₃) in the dark at 27-28° C. Embryogenic callusproliferating from the scutellum of the embryos is the transferred to CMmedia (N6 salts, Nitsch and Nitsch vitamins, 1 mg/l 2,4-D, Chu, et al.,(1985) Sci. Sinica 18:659-668). Callus cultures are maintained on CM byroutine sub-culture at two week intervals and used for transformationwithin 10 weeks of initiation.

Callus is prepared for transformation by subculturing 0.5-1.0 mm piecesapproximately 1 mm apart, arranged in a circular area of about 4 cm indiameter, in the center of a circle of Whatman #541 paper placed on CMmedia. The plates with callus are incubated in the dark at 27-28° C. for3-5 days. Prior to bombardment, the filters with callus are transferredto CM supplemented with 0.25 M mannitol and 0.25 M sorbitol for 3 hr inthe dark. The petri dish lids are then left ajar for 20-45 minutes in asterile hood to allow moisture on tissue to dissipate.

Each genomic DNA fragment is co-precipitated with pML18 containing theselectable marker for rice transformation onto the surface of goldparticles. To accomplish this, a total of 10 μg of DNA at a 2:1 ratio oftrait:selectable marker DNAs are added to 50 μl aliquot of goldparticles that have been resuspended at a concentration of 60 mg ml⁻¹.Calcium chloride (50 μl of a 2.5 M solution) and spermidine (20 μl of a0.1 M solution) are then added to the gold-DNA suspension as the tube isvortexing for 3 min. The gold particles are centrifuged in a microfugefor 1 sec and the supernatant removed. The gold particles are thenwashed twice with 1 ml of absolute ethanol and then resuspended in 50 μlof absolute ethanol and sonicated (bath sonicator) for one second todisperse the gold particles. The gold suspension is incubated at −70° C.for five minutes and sonicated (bath sonicator) if needed to dispersethe particles. Six μl of the DNA-coated gold particles are then loadedonto mylar macrocarrier disks and the ethanol is allowed to evaporate.

At the end of the drying period, a petri dish containing the tissue isplaced in the chamber of the PDS-1000/He. The air in the chamber is thenevacuated to a vacuum of 28-29 inches Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1080-1100 psi. Thetissue is placed approximately 8 cm from the stopping screen and thecallus is bombarded two times. Two to four plates of tissue arebombarded in this way with the DNA-coated gold particles. Followingbombardment, the callus tissue is transferred to CM media withoutsupplemental sorbitol or mannitol.

Within 3-5 days after bombardment the callus tissue is transferred to SMmedia (CM medium containing 50 mg/l hygromycin). To accomplish this,callus tissue is transferred from plates to sterile 50 ml conical tubesand weighed. Molten top-agar at 40° C. is added using 2.5 ml of topagar/100 mg of callus. Callus clumps are broken into fragments of lessthan 2 mm diameter by repeated dispensing through a 10 ml pipet. Threeml aliquots of the callus suspension are plated onto fresh SM media andthe plates are incubated in the dark for 4 weeks at 27-28° C. After 4weeks, transgenic callus events are identified, transferred to fresh SMplates and grown for an additional 2 weeks in the dark at 27-28° C.

Growing callus is transferred to RM1 media (MS salts, Nitsch and Nitschvitamins, 2% sucrose, 3% sorbitol, 0.4% gelrite+50 ppm hyg B) for 2weeks in the dark at 25° C. After 2 weeks the callus is transferred toRM2 media (MS salts, Nitsch and Nitsch vitamins, 3% sucrose, 0.4%gelrite+50 ppm hyg B) and placed under cool white light (˜40 μEm⁻²s⁻¹)with a 12 hr photo period at 25° C. and 30-40% humidity. After 2-4 weeksin the light, callus begin to organize, and form shoots. Shoots areremoved from surrounding callus/media and gently transferred to RM3media (½×MS salts, Nitsch and Nitsch vitamins, 1% sucrose+50 ppmhygromycin B) in phytatrays (Sigma Chemical Co., St. Louis, Mo.) andincubation is continued using the same conditions as described in theprevious step.

Plants are transferred from RM3 to 4″ pots containing Metro mix 350after 2-3 weeks, when sufficient root and shoot growth have occurred.The seed obtained from the transgenic plants is examined for geneticcomplementation of the ETO1 mutation with the wild-type genomic DNAcontaining the ETO1 gene.

Example 9 Variants of ETO1 Sequences

A. Variant Nucleotide Sequences of ETO1 Proteins that do not Alter theEncoded Amino Acid Sequence

The ETO1 sequences having the nucleotide sequence of the open readingframe with about 70%, 75%, 80%, 85%, 90% and 95% nucleotide sequenceidentity when compared to the starting unaltered ORF nucleotide sequenceof the corresponding SEQ ID NO: 1, 3, 5, 7 or 9. These functionalvariants are generated using a standard codon table. While thenucleotide sequence of the variants are altered, the amino acid sequenceencoded by the open reading frames do not change.

B. Variant Amino Acid Sequences of ETO 1 Polypeptides

Variant amino acid sequences of the ETO1 polypeptides are generated. Inthis example, one amino acid is altered. Specifically, the open readingframes are reviewed to determine the appropriate amino acid alteration.The selection of the amino acid to change is made by consulting theprotein alignment (with the other orthologs and other gene familymembers from various species). An amino acid is selected that is deemednot to be under high selection pressure (not highly conserved) and whichis rather easily substituted by an amino acid with similar chemicalcharacteristics (i.e., similar functional side-chain). Using the proteinalignment, an appropriate amino acid can be changed. Once the targetedamino acid is identified, the procedure outlined in the followingsection C is followed. Variants having about 70%, 75%, 80%, 85%, 90% and95% nucleic acid sequence identity are generated using this method.

C. Additional Variant Amino Acid Sequences of ETO1 Polypeptides

In this example, artificial protein sequences are created having 80%,85%, 90% and 95% identity relative to the reference protein sequence.This latter effort requires identifying conserved and variable regionsfrom the alignment and then the judicious application of an amino acidsubstitutions table. These parts will be discussed in more detail below.

Largely, the determination of which amino acid sequences are altered ismade based on the conserved regions among ETO1 protein or among theother ETO1 polypeptides. Based on the sequence alignment, the variousregions of the ETO1 polypeptide that can likely be altered arerepresented in lower case letters, while the conserved regions arerepresented by capital letters. It is recognized that conservativesubstitutions can be made in the conserved regions below withoutaltering function. In addition, one of skill will understand thatfunctional variants of the ETO1 sequence of the invention can have minornon-conserved amino acid alterations in the conserved domain.

Artificial protein sequences are then created that are different fromthe original in the intervals of 80-85%, 85-90%, 90-95% and 95-100%identity. Midpoints of these intervals are targeted, with liberallatitude of plus or minus 1%, for example. The amino acids substitutionswill be effected by a custom Perl script. The substitution table isprovided below in Table 3.

TABLE 3 Substitution Table Strongly Similar Rank of Amino and OptimalOrder to Acid Substitution Change Comment I L, V 1 50:50 substitution LI, V 2 50:50 substitution V I, L 3 50:50 substitution A G 4 G A 5 D E 6E D 7 W Y 8 Y W 9 S T 10 T S 11 K R 12 R K 13 N Q 14 Q N 15 F Y 16 M L17 First methionine cannot change H Na No good substitutes C Na No goodsubstitutes P Na No good substitutes

First, any conserved amino acids in the protein that should not bechanged is identified and “marked off” for insulation from thesubstitution. The start methionine will of course be added to this listautomatically. Next, the changes are made.

H, C and P are not changed in any circumstance. The changes will occurwith isoleucine first, sweeping N-terminal to C-terminal. Then leucine,and so on down the list until the desired target it reached. Interimnumber substitutions can be made so as not to cause reversal of changes.The list is ordered 1-17, so start with as many isoleucine changes asneeded before leucine, and so on down to methionine. Clearly many aminoacids will in this manner not need to be changed. L, I and V willinvolve a 50:50 substitution of the two alternate optimal substitutions.

The variant amino acid sequences are written as output. Perl script isused to calculate the percent identities. Using this procedure, variantsof the ETO1 polypeptides are generating having about 80%, 85%, 90% and95% amino acid identity to the starting unaltered ORF nucleotidesequence of SEQ ID NO: 1, 3, 5, 7 or 9.

Example 10 Transgenic Maize Plants

T₀ transgenic maize plants containing the ETO1 construct under thecontrol of a promoter are generated. These plants are grown ingreenhouse conditions, under the FASTCORN system, as detailed in USPatent Application Publication Number 2003/0221212, U.S. patentapplication Ser. No. 10/367,417.

Each of the plants is then analyzed for measurable alteration in one ormore of the following characteristics in the following manner:

T₁ progeny derived from self fertilization of each T₀ plant containing asingle copy of each construct that were found to segregate 1:1 for thetransgenic event were analyzed for improved growth rate in low KNO₃.Growth is monitored up to anthesis when cumulative plant growth, growthrate and ear weight were determined for transgene positive, transgenenull, and non-transformed controls events. The distribution of thephenotype of individual plants was compared to the distribution of acontrol set and to the distribution of all the remaining treatments.Variances for each set were calculated and compared using an F test,comparing the event variance to a non-transgenic control set varianceand to the pooled variance of the remaining events in the experiment.The greater the response to KNO₃, the greater the variance within anevent set and the greater the F value. Positive results will be comparedto the distribution of the transgene within the event to make sure theresponse segregates with the transgene.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. A method for reducing ethylene biosynthesis ina plant, comprising: (a) introducing into a plant cell a recombinantexpression cassette comprising a first polynucleotide operably linked toa heterologous promoter, wherein said expression cassette directsdown-regulation of a second polynucleotide, wherein the secondpolynucleotide is selected from the group consisting of: i. apolynucleotide comprising the full length nucleotide sequence of SEQ IDNO: 1, 3, 5, 7 or 9; ii. a polynucleotide having at least 95% sequenceidentity, as determined by the BLAST 2.0 algorithm under defaultparameters, to the full length of the sequence set forth in SEQ ID NO:1, 3, 5, 7 or 9, wherein the polynucleotide encodes a polypeptide havingETO1 activity; and iii. a polynucleotide encoding a polypeptidecomprising the full length amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8 or 10; (b) culturing the plant cell under plant cell growingconditions and regenerating a plant therefrom; and (c) inducingexpression of said polynucleotide for a time sufficient to reduce thelevel of ethylene biosynthesis in said plant.
 2. The method of claim 1,wherein the first polynucleotide is operably linked to the heterologouspromoter in antisense orientation.
 3. The method of claim 1, wherein theplant cell is from a plant selected from the group consisting of maize,soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice,barley and millet.
 4. The method of claim 3, wherein the plant is maize.5. The method of claim 1, wherein the construct is an over-expressionconstruct.
 6. The method of claim 1, wherein the first polynucleotidecomprises a nucleotide sequence selected from the group consisting of:(a) the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9; (b)a polynucleotide amplified from a nucleic acid library using primerswhich selectively hybridize, under stringent hybridization conditions,to loci within a polynucleotide having the sequence set forth in SEQ IDNO: 1, 3, 5, 7 or 9, wherein the amplified polynucleotide encodes apolypeptide comprising ETO1 activity; (c) a polynucleotide at least 25bases in length which is identical to the corresponding portion of SEQID NO: 1, 3, 5, 7, or 9; (d) a polynucleotide encoding a polypeptidehaving at least 95% sequence identity to the full length of SEQ ID NO:2, 4, 6, 8 or 10, wherein the polynucleotide encodes a polypeptidecomprising ETO1 activity; and (e) a polynucleotide which is thefull-length complement of a polynucleotide of (a), (b), (c), or (d). 7.A transgenic plant produced by the method of claim
 1. 8. The transgenicplant of claim 7, wherein the plant has decreased ethylene productionwhen compared to a control plant.
 9. A recombinant expression cassette,comprising a polynucleotide operably linked to a heterologous promoter,wherein said polynucleotide is selected from the group consisting of:(a) a polynucleotide comprising the full length nucleotide sequence ofSEQ ID NO: 1, 3, 5, 7 or 9; (b) a polynucleotide having at least 95%sequence identity, as determined by the BLAST 2.0 algorithm underdefault parameters, to the full length of the sequence set forth in SEQID NO: 1, 3, 5, 7 or 9, wherein the polynucleotide encodes a polypeptidehaving ETO1 activity; (c) a polynucleotide encoding a polypeptidecomprising the full length amino acid sequence set forth in SEQ ID NO:2, 4, 6, 8 or 10; (d) a polynucleotide amplified from a nucleic acidlibrary using primers which selectively hybridize, under stringenthybridization conditions, to loci within a polynucleotide having thesequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9, wherein the amplifiedpolynucleotide encodes a polypeptide comprising ETO1 activity; (e) afirst polynucleotide which selectively hybridizes, under stringenthybridization conditions and a wash in 2×SSC at 50° C., to a secondpolynucleotide selected from the group consisting of the nucleotidesequences set forth in SEQ ID NO: 1, 3, 5, 7 and 9, wherein the firstpolynucleotide encodes a polypeptide comprising ETO1 activity; (f) apolynucleotide at least 25 bases in length which is identical to thecorresponding portion of SEQ ID NO: 1, 3, 5, 7, or 9; (g) apolynucleotide encoding a polypeptide having at least 95% sequenceidentity to the full length of SEQ ID NO: 2, 4, 6, 8 or 10, wherein thepolynucleotide encodes a polypeptide comprising ETO1 activity; and (h) apolynucleotide which is the full-length complement of a polynucleotideof (a), (b), (c), (d), (e), (f), or (g).
 10. The recombinant expressioncassette of claim 9, wherein the polynucleotide is operably linked tothe heterologous promoter in antisense orientation.
 11. Therecombination expression cassette of claim 9, wherein the heterologouspromoter is a tissue preferred promoter.